Biomarkers for non-hodgkin lymphomas and uses thereof

ABSTRACT

The disclosure provides a method of identifying a subject as having B-cell non-Hodgkin lymphoma (NHL) such as testing a sample from a subject for a mutation in one or more biomarkers. Also described are methods of classifying or monitoring a subject having, or suspected of having, B-cell non-Hodgkin lymphoma comprising testing the same for a mutation in one or more biomarkers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 13/805,504, filed on Dec. 19, 2012, which is a U.S. national phase application filed under 35 U.S.C. § 371 claiming benefit to International Patent Application No. PCT/CA2011/000724, filed on Jun. 23, 2011, which in turn claims benefit pursuant to 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 61/357,813 filed Jun. 23, 2010 and U.S. Provisional Application No. 61/420,065 filed Dec. 6, 2010, each of which application is hereby incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates to methods of testing for cancer and more specifically to methods of testing samples for somatic mutations indicative of B-cell Non-Hodgkin Lymphomas (NHLs).

BACKGROUND OF THE DISCLOSURE

Non-Hodgkin lymphomas (NHLs) are cancers of B, T or natural killer lymphocytes. The two most common types of NHL, follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), together comprise 60% of new B-cell NHL diagnoses each year in North America [1]. FL is an indolent and typically incurable disease characterized by clinical and genetic heterogeneity. DLBCL is aggressive and likewise heterogeneous, comprising at least two distinct subtypes that respond differently to standard treatments. Both FL and the germinal centre B-cell (GCB) cell of origin (COO) subtype of DLBCL derive from germinal centre B cells whereas the activated B-cell (ABC) variety, which exhibits a more aggressive clinical course, is thought to originate from B cells that have exited, or are poised to exit, the germinal centre [2]. Current knowledge of the specific genetic events leading to DLBCL and FL is limited to the presence of a few recurrent genetic abnormalities [2]. For example, 85-90% of FL and 30-40% of GCB DLBCL cases [3, 4] harbour t(14;18)(q32;q21), which results in deregulated expression of the BCL2 oncoprotein. Other genetic abnormalities unique to GCB DLBCL include amplification of the c-REL gene and of the miR-17-92 microRNA cluster [5]. In contrast to GCB cases, 24% of ABC DLBCLs harbour structural alterations or inactivating mutations affecting PRDM1, which is involved in differentiation of GCB cells into antibody-secreting plasma cells [6]. ABC-specific mutations also affect genes regulating NF-κB signalling [7-9], with TNFAIP3 (A20) and MYD88 [10] the most abundantly mutated in 24% and 39% of cases respectively.

Despite the disparity in response to therapy of the individual subtypes and the knowledge of clear genetic differences between the subtypes, clearly identifying B-cell NHLs remains challenging. Accordingly, there is a need for improved methods of identifying as well as classifying B-cell NHLs including GCB and ABC DLBCLs.

SUMMARY OF THE DISCLOSURE

In one aspect, the present disclosure is directed towards new and useful methods for the identification and/or classification of B-cell NHLs. As described herein, the inventors have (1) identified somatic mutations and (2) determined the prevalence, expression and focal recurrence of mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) in order clarify the genetic architecture of B-cell NHLs. Using strategies and techniques applied to cancer genome and transcriptome characterization [11-13], tumour DNA and/or RNA was sequenced from 117 tumour samples and 10 cell lines and 651 genes were identified with evidence of somatic mutation in B-cell NHL. After validation, 109 genes were shown to be somatically mutated in 2 or more NHL cases. The frequency and nature of mutations within MLL2 and MEF2B, which were among the most frequently mutated genes with no previously known role in lymphoma are also described herein.

As set out in Example 1, a number of biomarkers useful for identifying samples with B-cell NHL have been identified. More specifically, the biomarkers listed in Table 1 have been confirmed as somatic mutations in tumour samples from subjects with B-Cell NHL and show significant evidence for positive selection. In another aspect of the disclosure, a number of biomarkers useful for classifying samples into subtypes of B-cell NHLs have been identified. Some biomarkers have been shown to be selectively mutated in either germinal centre B-cell (GCB) Diffuse Large B-cell Lymphoma (DLBCL) or Activated B-Cell (ABC) DLBCL and are therefore useful for classifying samples as belonging to either the GCB or ABC subtype of DLBCL. Thus, application of the methods described herein allows for the identification of those subjects with specific subtypes of B-cell NHL and enable improved disease management and pharmacological treatment with agents best suited to a particular disease subtype.

Remarkably, a number of the biomarkers associated with B-cell NHLs described herein are involved in histone modification. More specifically, the inventors have discovered that at least five biomarkers (MLL2, MEF2B, CREBBP, EP300, EZH2 and HDAC7) shown to be selectively mutated in B-cell NHLs are predicted to be involved in the process of histone modification. Post-translational modifications of histones, such as methylation and acetylation, can affect the accessibility of stretches of genomic DNA to transcription factors. Mutations in MLL2 are predicted to affect levels of histone methylation while mutations in MEF2B are predicted to affect histone acetylation. Moreover, mutations in MEF2B are predicted to affect the ability of MEF2B to regulate acetylation levels via these three enzymes (HDAC7, CREBBP and EP300). Testing a sample for mutations in histone modifying genes is therefore useful for the identification of B-cell NHLs.

Accordingly, in one aspect there is provided a method of identifying a subject as having B-cell non-Hodgkin lymphoma (NHL), the method comprising testing a sample from the subject for a mutation in one or more biomarkers listed in Table 1. In one embodiment, the presence of a mutation in the sample identifies the subject as having B-cell NHL. In one embodiment, the method comprises detecting one or more mutations in a nucleic acid molecule coding for a biomarker. In one embodiment, the method comprises detecting one or more mutations in a polypeptide or protein coding for a biomarker. In one embodiment, the method comprises detecting mutations in one or more histone modifying genes such as MLL2, MEF2B, CREBBP, EP300, EZH2 or HDAC7. In one embodiment, the biomarkers are selected from FOX01, CCND3, BTG2, B2M, TNFRS14, CREBBP, EP300, BCL10, BTG1, GNA13, SGK1, MLL2, MEF2B, CD79B and MYD88. Optionally, 2 or more, 3 or more, 4 or more, 5 or more or greater than 5 of the biomarkers listed in Table 1 or described herein are tested for mutations. The methods described herein also include testing the sample for one or more of the mutations described herein such as those listed in Table 3, Table 5, Table 6, Table 7 or Table 9. In one embodiment, the biomarker is MEF2B and the method comprises detecting a mutation in a nucleic acid molecule or polypeptide corresponding to a mutation at amino acid position K4, Y69, N81 or D83 of the MEF2B polypeptide.

In another aspect of the disclosure, there is provided a method of classifying a subject suspected of having, or having, B-cell non-Hodgkin lymphoma (NHL). In one embodiment, the method comprises testing a sample from the subject for a mutation in one or more biomarkers selected from MEF2B, SGK1, GNA13, and TNFRS14. In one embodiment, samples that have one or more mutations in one or more biomarkers selected from MEF2B, SGK1, GNA13, and TNFRS14 are classified as having germinal centre B-cell (GCB) Diffuse Large B cell lymphoma (DLBCL). Optionally, the method further comprises testing the sample for a mutation in BCL2, TP53 or EZH2.

In one aspect of the disclosure, there is provided a method of classifying a subject suspected of having, or having, B-cell non-Hodgkin lymphoma (NHL) comprising testing the sample for one or more mutations in MYD88 or CD79B. In one embodiment, samples that have a mutation in MYD88 or CD79B are classified as having activated B-cell (ABC) Diffuse Large B cell lymphoma. Optionally, the method for classifying a subject suspected of having, or having, B-cell non-Hodgkin lymphoma (NHL) includes testing for one or more of MEF2B, SGK1, GNA13, TNFRS14, MYD88 or CD79B.

The methods described herein are also useful for classifying a subject in order to select a suitable treatment for the subject. In one embodiment, the methods include selecting a treatment for a subject based on the classification of the sample as GCB DLBCL or ABC DLBCL. For example, in one embodiment the sample is classified as GCB DLBCL, and a treatment is selected that comprises administration of a histone deacetylase (HDAC) inhibitor-class drug. In one embodiment, the methods for classifying a subject described herein comprise testing a sample from the subject for one or more of the mutations listed in Table 3, Table 5, Table 6, Table 7 or Table 9.

In another aspect of the disclosure, there is provided a method of monitoring a subject with B cell non-Hodgkin lymphoma (NHL) comprising testing a first sample from the subject for a mutation in one or more biomarkers listed in Table 1 and comparing the results to a control. Optionally, the control represents results from testing a second sample taken from the subject at an earlier time point. In one embodiment, the method comprises testing one or more biomarkers selected from MLL2, MEF2B, CREBBP, EP300, EZH2, H3K27, FOX01, CCND3, BTG2, B2M, TNFRS14, BCL10, BTG1, GNA13, SGK1, MYD88 and CD79B. In one embodiment, the method comprises testing for one or more of the mutations listed in Table 3, Table 5, Table 6, Table 7 or Table 9.

Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments of the present disclosure will now be described in relation to the drawings in which:

FIG. 1 shows a genome-wide visualization of somatic mutation targets in NHL. Overview of structural rearrangements and copy number variations (CNVs) in the 11 DLBCL genomes and protein-altering single nucleotide variants (coding SNVs; cSNVs) in the 109 recurrently mutated genes identified in our analysis. Inner arcs represent somatic fusion transcripts identified in one of the genomes. The CNVs (copy number variants) and LOH (loss of heterozygosity) detected in each of the 11 DLBCL tumour/normal pairs are displayed on the concentric sets of rings. The inner 11 rings show regions of enhanced homozygosity plotted with blue (interpreted as LOH). The outer 11 rings show somatic CNVs. Purple circles indicate the position of genes with at least two confirmed somatic mutations with circle diameter proportional to the number of cases with cSNVs detected in that gene. Circles representing the genes with significant evidence for positive selection are labeled. Coincidence between recurrently mutated genes and regions of gain/loss are colour-coded in the labels (green=loss, red=gain). For example B2M, which encodes beta-2-microglobulin, is recurrently mutated and is deleted in two cases.

FIG. 2 shows an overview of mutations and potential cooperative interactions in NHL. This heat map displays possible trends towards co-occurrence (red) and mutual exclusion (blue) of somatic mutations and structural rearrangements. Colours were assigned by taking the minimum value of a left- and right-tailed Fisher exact test. To capture trends a P-value threshold of 0.3 was used, with the darkest shade indicating those meeting statistical significance (P<=0.05). The relative frequency of mutations in ABC (dark grey), GCB (darkest grey), unclassifiable (light grey) DLBCLs and FL (lightest grey) cases is shown on the left. Genes were arranged with those having significant (P<0.05, Fisher exact test) enrichment for mutations in ABC cases (dark grey triangle) towards the top (and left) and those with significant enrichment for mutations in GCB cases (darkest grey triangle) towards the bottom (and right). The total number of cases in which each gene contained either cSNVs or confirmed somatic mutations is shown at the top. The cluster of squares (upper-right) results from the mutual exclusion of the ABC-enriched mutations (e.g. MYD88, CD79B) from the GCB-enriched mutations (e.g. EZH2, GNA13, MEF2B, SGK1). Presence of structural rearrangements involving the two oncogenes BCL6 and BCL2 (indicated as BCL6s and BCL2s) was determined with FISH techniques utilizing break-apart probes.

FIG. 3 shows a summary and effect of somatic mutations affecting MLL2 and MEF2B. (A) Re-sequencing the MLL2 locus in 89 samples revealed mainly nonsense (dark grey circles) and frameshift-inducing indel mutations (triangles). A smaller number of non-synonymous somatic mutations (light grey circles) and point mutations or deletions affecting splice sites (stars) were also observed. All of the non-synonymous point mutations affected a residue within either the catalytic SET domain, the FYRC domain (“FY-rich C-terminal domain”) or PHD zinc finger domains. The effect of these splice site mutations on MLL2 splicing was also explored. (B) The cSNVs and somatic mutations found in MEF2B in all FL and DLBCL cases sequenced are shown with the same symbols. Only the amino acids with variants in at least two patients are labelled. cSNVs were most prevalent in the first two protein coding exons of MEF2B (exons 2 and 3). The crystal structure of MEF2 bound to EP300 supports that two of the four hot spots (N67 and Y69) are important in the interaction between these proteins [50].

FIG. 4 shows the N-terminal truncation of FOXO1 protein with mutation affecting initial codon. (A) The RNA-seq data of cell lines and patient samples revealed mutations in 3 samples affecting the initial codon of FOXO1. To determine the effect of such mutations on FOXO1 protein, we assayed FOXO1 by Western blot in DLBCL cell lines using an antibody raised against full-length FOXO1 (2H8.2). In the cell line containing a mutation at the initiator methionine (OCI-Ly1), we observed a FOXO1 band of reduced molecular weight, compared to FOXO1 wild-type cell lines (size indicated in Kilodaltons on the left). The reduced size is consistent with the use of a second methionine codon in the FOXO1 gene, producing a protein shortened at the amino terminus by 70 amino acids. The same blot was also probed with an antibody that recognizes an N-terminal epitope (L27) and lack of a band in OCI-Ly1 cells is consistent with the notion that the lower band in this cell line corresponds to FOXO1 protein lacking its N-terminus. Absence of the protein in the DB cell line was noted, which showed significantly reduced mRNA levels as measured by RNA-seq (upper bar chart; RPKM=Reads Per Kilobase of gene model per Million mapped reads).

FIG. 5 shows the effect of GNA13 mutations at the protein level. (A) A western blot revealed the expected lack of GNA13 protein in DOHH2, the cell line with a truncating point mutation detected in the RNA-seq data. The lack of protein in Karpas422, SU-DHL-6 and WSU-DLCL2 was surprising, as protein-truncating mutations were not detected in these cells. (B) Further analysis of the aligned sequence from these three cell lines and additional analysis utilizing a de-novo transcript assembly approach (Trans-ABySS; Methods), revealed multiple aberrations that may explain the lack of protein. Firstly, in Karpas422 reads were observed to map the first intron, suggesting that the intron is retained in a significant proportion of GNA13 transcripts (compare Karpas422 on the left to WSU-DLCL2 on the right). Inspection of sequence contigs from this case revealed the likely cause of intron reads to be a deletion of 87 nt that removes the canonical splicing donor from this exon (Panel C, top). Splicing still appears to occur to a lesser extent using a non-GT donor. Assembled reads from SU-DHL-6 revealed a 2 nt deletion and a large 1028 nt deletion. The former would affect the reading frame and the latter removes the terminal stop codon. Finally, in WSU-DLCL2, the splicing donor after the third exon was apparently mutated, converting the GT donor to a GC sequence (not shown). As in the Karpas422 case, there was clear evidence for retention of this intron in GNA13 transcripts in WSU-DLCL2. Intron retention has previously been linked to nonsense-mediated transcript degradation [76] and if that is the case here, could explain the lack of GNA13 protein in these cells.

FIG. 6 shows the predicted impact of recurrently mutated genes on BCR signalling and downstream messengers. (A) Autocrine and paracrine stimulation of IL-21R induces the dimerization and activation of STAT3, a positive regulator of PRDM1 expression [77]. Mutations affecting the DNA binding domain of STAT3 are known to act as dominant negatives, which would predict the inability to induce PRDM1 expression following IL-21 stimulation. (B) Multiple mutations predicted to directly alter BCR signalling or alter the normal events subsequent to BCR-induced influx of the secondary messenger Ca²⁺. Cross-linking of CD58 has been shown to result in the phosphorylation of BLNK, Syk and PLC-gamma and lead to Akt activation [78]. Various mutations are expected to alter the ability of B cells to induce the expression of MEF2 target genes in response to the Ca²⁺ influx. The role of MEF2 gene family members in mediating epigenetic alterations downstream of the BCR has been inferred from a knockout study in which MEF2C was shown to be required for mediating calcium-dependent response to BCR signaling [79] and the involvement of CREBBP/EP300 in this process has been inferred from MEF2-mediated transcriptional regulation in other cell types including T cells [80]. This model predicts that influx of Ca²⁺ after BCR stimulation would result in the displacement of HATs by activated Calmodulin-dependent protein kinase (CAMK), allowing HDAC activity via CREBBP/EP300 thus enabling transcription at MEF2 target loci. In this model, mutation of any of these three genes and potentially the S155F mutation in HDAC7 would diminish this effect and suppress the induction of MEF2 target loci after BCR stimulation. (C) Multiple mutations may affect the regulation of the activity of FOXO proteins following BCR stimulation. FOXO1 is a downstream target of the kinase AKT, which is activated during BCR signalling. SGK, a related kinase (mutated in B-cell NHLs as described herein), is known to phosphorylate FOXO3a in a similar way [25] and the present applicants predict it to also phosphorylate FOXO1. Thus, mutations affecting the FOXO1 phosphorylation site or SGK1 could affect the regulation of FOXO1 nuclear localization and hence, its transactivation activity. The shortened FOXO1 protein produced by mutation of the initial codon (FIG. 4) would not contain this phosphorylation site and hence those mutations may also result in altered subcellular localization. Various mutations affecting NF-κB activity, which have been previously described, were also observed here [9-10, 18, 21]. (D) Many of the recurrently mutated genes in B-NHL are involved in histone modification or themselves encode histone proteins (i.e. HIST1H1C, one of multiple genes that encode histone protein H1). CREBBP/EP300 and MLL2 each produce activating chromatin marks (H3K27Ac and H3K4me3, respectively). HDAC (e.g. HDAC7) and EZH2 produce inactivating marks by removing acetyl groups and trimethylating H3K27, respectively. As heterozygous EZH2 Y641 mutations are known to effectively enhance PRC2 activity [43], then each of the individual mutations may result in suppression of gene expression. It have not been confirmed whether EZH2 and MLL2 regulate the expression of the same genes as MEF2B/CREBBP/EP300.

DETAILED DESCRIPTION OF THE DISCLOSURE I. Definitions

As used herein, “B-cell Non-Hodgkin Lymphoma” or “B-cell NHL” refers to any lymphoma of B-cells except those classified as Hodgkin lymphoma. As used herein, “lymphoma” refers to a cancer in the lymphatic cells of the immune system.

As used herein, “follicular lymphoma” or “FL” refers to a lymphoma of follicle center B-cells (centrocytes and centroblasts), which has at least a partially follicular pattern.

As used herein, “Diffuse Large B cell lymphoma” or “DLBCL” refers to a lymphoma of B-cells wherein the cells are generally about 4-5 times the diameter of small lymphocytes and typically have marked cell-to-cell variation in size and shape. Typically, their cytoplasm is basophilic and moderate in abundance. Nucleoli can be small but conspicuous to large and prominent and may be peripheral and/or central.

As used herein “germinal centre B-cell lymphoma” or “GCB lymphoma” refers to a subtype of DLBCL wherein the lymphoma appears to arise from germinal centre B cells. Typically, GCB cells have a pattern of genetic expression that is similar to germinal center B cells and often a chromosomal translocation involving the gene bcl-2.

As used herein “activated B-Cell lymphoma” or “ABC lymphoma” refers to a subtype of DLBCL wherein the lymphoma appears to arise from postgerminal centre B cells that are arrested during plasmacytic differentiation.

The term “biomarker” as used herein can be any type of molecule corresponding to a gene listed in Table 1, or any type of molecule identified herein which can be used to distinguish samples with or without B-cell NHL or between subtypes of B-cell NHL. The term biomarker includes without limitation, a nucleic acid sequence including a gene, or corresponding RNA or cDNA, or a polypeptide, fragment thereof, or epitope that is differentially present, including differentially modified (e.g. differentially glycosylated), expressed, and/or soluble biomarkers e.g. biomarkers which are detectable in a biological fluid and which are differentially cleaved, secreted, released or shed in subjects with or without B-cell NHL. In one embodiment, detecting one or more mutations in one or more biomarkers in a sample from a subject indicates that the subject has B-cell NHL.

As used herein, the term “sample” refers to any biological fluid, cell or tissue sample from a subject which can be assayed for biomarkers (e.g. DNA, RNA and/or polypeptide products), such as soluble biomarkers in subjects having or not having B-cell NHL. Optionally, the sample comprises nucleic acids and/or proteins that have been isolated, purified or otherwise treated. For example, a sample may be fractionated (e.g. by centrifugation or using a column for size exclusion), concentrated or proteolytically processed such as trypsinized, depending on the method of testing for mutations in the biomarker employed. The sample may be a biological fluid such as blood, serum, saliva, cerebrospinal fluid, plasma, or lymphatic fluid, a tissue sample or tissue biopsy. In one embodiment, the sample is a “tumour sample”. As used herein “tumour sample” refers to a sample of cells from a subject that is undergoing uncontrolled cell division. In a preferred embodiment, the sample comprises all or part of one or more lymphoid cells, lymph nodes or a lymph node biopsy. In another preferred embodiment, the sample is a blood sample or plasma sample.

As used herein, the term “subject” refers to any member of the animal kingdom, and includes mammals such as humans. The term also includes subjects having cancer or suspected of having cancer, such as B-cell NHL. Optionally, the subject is symptomatic or asymptomatic of B-cell NHL.

As used herein the phrase “subject suspected of having B-cell non-Hodgkin lymphoma” refers to a subject for which information regarding whether or not the subject has B-cell NHL or a particular subtype of B-cell NHL is desired. Optionally, a subject suspected of having B-cell NHL may present with one or more symptoms such as: swollen, painless lymph nodes in the neck, armpits, or groin; sudden weight loss; coughing, trouble breathing, or chest pain; and/or pain or swelling in the abdomen.

As used herein “mutation” refers to a variant of biomarker that does not appear in a control sample that alters the presence, amount or biological activity of a biomarker as described herein. In one embodiment the control sample is from a subject that does not have B-cell NHL or from a sample that is not undergoing uncontrolled cell division. In one embodiment, the control sample is from the same subject as the test subject but is taken at a different point in time. In one embodiment, the mutation is a variant of the wild-type nucleic acid sequence or polypeptide sequence for that biomarker. In one embodiment, the mutation is a nonsense mutation, non-synonymous mutation, insertion or deletion. In one embodiment, the mutation is not known prior to testing the sample for a mutation. In one embodiment, the mutation is a coding Single Nucleotide Variant (cSNV). In one embodiment, the mutation is a copy number variant (CNV) or loss of heterozygozity (LOH). As used herein, the term “somatic mutation” refers to a mutation that is acquired after the formation of a zygote and is not found in the majority of cells in a subject. Examples of mutations include those listed herein in Tables 3, 5, 6, 7 and 9.

As used herein “testing a sample from the subject for a mutation” refers to analyzing the sample to determine the presence or absence of a mutation in a biomarker. In one embodiment, testing the sample for a mutation involves sequencing nucleic acid molecules that encode the biomarker or part of the biomarker. In another embodiment, testing the sample for a mutation involves detecting a mutant polypeptide such as by protein sequencing, use of selective antibodies, or the use of mass spectrometry based genotyping assays.

As used herein, “classifying a subject as having germinal centre B-cell lymphoma” refers to identifying the subject as being more likely to have germinal centre B-cell lymphoma than other types of B-cell NHL. In one embodiment, a subject classified as having GCB lymphoma is excluded from having ABC lymphoma.

As used herein, “classifying a subject as having activated B-cell lymphoma” refers to identifying the subject as being more likely to have Activated B-cell lymphoma than other types of B-cell NHL. In one embodiment, a subject classified as having ACB lymphoma is excluded from having GCB lymphoma.

As used herein “selecting a treatment” refers to determining a course of therapeutic action for a subject from a plurality of possible treatment options. For example, “selecting a treatment” may comprise selecting a specific pharmaceutical agent for administration to a subject with B-cell NHL in need thereof, as opposed to another pharmaceutical agent which may be ineffective for a particular subtype of B-cell NHL. Clinical trials that test the selective activity of therapies in ABC DLBCL are ongoing. These include the utility of drugs that reduce the activity of the transcription factor NFkB, thus reducing expression of NFkB target genes. Such drugs include Bortezomib and Lenalidomide[100; 101].

As used herein, “monitoring a subject with B-cell non Hodgkin lymphoma” refers to ascertaining the progression or remission of the B-cell NHL in a subject over time.

II. Methods

Methods for Identifying B-Cell NHLs

The present disclosure pertains to methods for detecting B-cell NHLs using biomarkers that have been shown to be mutated in samples from subjects with B-Cell NHL. As set out in Example 1, the biomarkers identified in Table 1 have been shown to be mutated in at least 2 or more cases of NHL and furthermore exhibit evidence for positive selection with either selective pressure for acquiring non-synonymous point mutations or truncating/nonsense mutations.

Accordingly, in one embodiment, there is provided a method of identifying a subject as having B-cell non-Hodgkin lymphoma comprising testing a sample from the subject for a mutation in one or more biomarkers listed in Table 1. A variety of methods known in the art may be used to test the sample to identify mutations in the biomarkers. For example, mutations may be detected in a nucleic acid molecule such as genomic DNA or mRNA. Alternatively, mutations may be detected in a polypeptide that corresponds to a biomarker listed in Table 1. In one embodiment, the mutation is listed in Tables 3, 5, 6, 7 or 9. In a preferred embodiment, the sample is tested for mutations by sequencing DNA coding for the biomarker. Optionally, the method involves amplifying the nucleic acid coding for the biomarker using PCR.

Various methods or techniques for identifying mutations in nucleic acid molecules that known in the art may be used in order to detect mutations in the biomarkers described herein. For example, embodiments include, but are not limited to, techniques such as primer extension, classical microarrays, sequencing or line probes. Methods of PCR product endpoint detection including, but not limited to, fluorescence, chemiluminescence, colourimetric techniques or measurement of redox potential may also be used with the embodiments described herein for detecting mutations in nucleic acid sequences. Optionally, the relative or absolute amount of a nucleic acid molecule corresponding to a biomarker is determined and compared to a control sample.

In another embodiment, various methods or techniques for identifying mutations in polypeptides that are known in the art may be used in order to detect mutations in the biomarkers described herein. For example, methods useful for detecting a mutation in a polypeptide corresponding to a biomarker as described herein, include mass spectrometry approaches, such as multiple reaction monitoring (MRM) and product-ion monitoring (PIM), and immunoassays such as Western blots, enzyme-linked immunosorbant assays (ELISA), and immunoprecipitation followed by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) immunocytochemistry and protein sequencing methods.

In one embodiment, antibodies or antibody fragments are used to detect a polypeptide of one or more biomarkers of the disclosure or the mutated forms a polypeptide of one or more biomarkers of the disclosure. Antibodies having specificity for a specific polypeptide, or a specific mutated polypeptide, such as the protein product of a biomarker gene of the disclosure, may be prepared by conventional methods. In an embodiment, the antibody or antibody fragment is labeled with a detectable marker. In a further embodiment, the antibody or antibody fragment is, or is derived from, a monoclonal antibody. A person skilled in the art will be familiar with the procedure for detecting the a polypeptide biomarker by using said antibodies or antibody fragments, for example, by contacting the sample from the subject with an antibody or antibody fragment labeled with a detectable marker, wherein said antibody or antibody fragment forms a complex with the biomarker. Optionally, the relative or absolute amount of a polypeptide corresponding to a biomarker is determined and compared to a control sample.

In one embodiment, the sample is from a subject having, or suspected of having, B-cell non-Hodgkin lymphoma. For example, in one embodiment the sample is a tumour sample from a subject with lymphoma. In one embodiment, the sample is a tumour biopsy of lymphoid tissue.

In one embodiment, the method comprises testing the sample for mutations in one or more biomarkers listed in Table 1. In one embodiment, the method comprises testing the sample for a plurality of the biomarkers listed in Table 1. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 15 or more of the biomarkers may be tested for mutations.

In one embodiment the method comprises testing one or more histone modifying genes. For example, in one embodiment the method comprises testing one or more of MLL2, MEF2B, CREBBP, EP300, EZH2 or H3K27. In one embodiment, the method comprises testing one or more of FOX01, CCND3, BTG2 and B2M. In one embodiment, the method comprises testing one or more of BTG1, GNA13, SGK1, MLL2 and MEF2B. In one embodiment, the method comprises testing one or more of EZH2, TNFRS14, CREBP, BCL10, BTG1, GNA13, SGK1, MLL2 and MEF2B.

Methods for Classifying B-Cell NHLs

In another aspect of the disclosure there is provided a method of classifying a subject suspected of having, or having, B-cell non-Hodgkin lymphoma (NHL) comprising testing the sample for a mutation in one or more biomarkers selected from MEF2B, SGK1, GNA13, and TNFRS14. In one embodiment, samples that have a mutation in MEF2B, SGK1, GNA13, or TNFRS14 are classified as having germinal centre B-cell (GCB) diffuse large B cell lymphoma (DLBCL). Optionally, the method further comprises testing the sample for mutations in additional genes known to be mutated in GCB such as BCL2, TP53 or EZH2. Optionally, the method comprises testing the sample for mutations in one or more the biomarkers listed in Table 1. Optionally, the method comprises testing the sample for one or more of the mutations listed in Tables 3, 5, 6, 7 or 9.

In another embodiment, there is provided a method of classifying a subject having, or suspected of having, B-cell non-Hodgkin lymphoma (NHL) comprising testing a sample from the subject for a mutation in MYD88 or CD79B. In one embodiment, samples that have a mutation in MYD88 or CD79B are classified as having activated B-cell (ABC) diffuse large B cell lymphoma. Optionally, the method comprises testing the sample for mutations in one or more the biomarkers listed in Table 1. Optionally, the method comprises screening the sample for one or more of the mutations listed in Tables 3, 5, 6, 7 or 9.

Classifying subjects with B-cell NHL into subtypes provides a more specific clinical diagnosis and facilitates selecting therapeutic treatment options for patients. In one embodiment, the methods described herein can be used to select a treatment for the subject based on the classification of a sample form the subject as GCB DLBCL or ABC DLBCL. For example, in one embodiment, subjects are classified as having germinal centre B-cell (GCB) diffuse large B cell lymphoma (DLBCL) and the treatment that is selected comprises administration of a histone deacetylase (HDAC) inhibitor-class drugs.

In another embodiment, the methods described herein can be used to monitor a subject with B-cell NHL. For example, in one embodiment the biomarkers described herein can be used to test a first sample from a subject and compare the results to a second sample taken from the subject at an earlier or later time point. In one embodiment, an increase in the number of mutations in the biomarkers described herein over time indicates a progression or worsening of the disease in the subject. In one embodiment, a reduction in the number of mutations in the biomarkers described herein over time indicates an improvement or remission of the disease in the subject. Optionally, one or more of the biomarkers listed in Table 1, or any combination thereof, can be tested in the methods for identifying, classifying or monitoring a subject as described herein.

While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Sequences associated with accession numbers or other identifiers described herein including for example the Tables and Figures, are herein specifically incorporated by reference.

The following non-limiting example is illustrative of the present disclosure:

Example 1 Identification of Recurrently Mutated Genes

The genomes or exomes of 14 NHL cases were sequenced, all with matched constitutional DNA sequenced to comparable depths. After screening for single nucleotide variants followed by subtraction of known polymorphisms and visual inspection of the sequence read alignments, 717 nonsynonymous (coding single nucleotide variants; cSNVs) affecting 651 genes were identified. Between 20 and 135 cSNVs in each of these genomes were identified. Only 25 of the 651 genes with cSNVs were represented in the cancer gene census (December, 2010 release) [14].

RNA sequencing (RNA-seq) was performed on these 14 NHL cases and an expanded set of 113 samples comprising 83 DLBCL, 12 FL and 8 B-cell NHL cases with other histologies and 10 DLBCL-derived cell lines. These data were analysed to identify novel fusion transcripts and cSNVs (FIG. 1). 240 genes were identified with at least one cSNV in a genome/exome or an RNA-seq “mutation hot spot” (below), and with cSNVs in at least three cases in total. cSNVs were selected from each of these 240 genes for re-sequencing to confirm their somatic status. Genes with previously documented mutations in lymphoma (e.g. CD79B, BCL2) were not re-sequenced. The somatic status of 543 cSNVs in 317 genes was confirmed, with 109 genes having at least two confirmed somatic mutations. A selection of these mutations is presented for biomarkers for B-cell NHL in Table 3. Of the successfully re-sequenced cSNVs predicted from the genomes, 171 (94.5%) were confirmed somatic, 7 were false calls and 3 were present in the germ line. These 109 recurrently mutated genes were significantly enriched for genes implicated in lymphocyte activation (P=8.3×10⁻⁴; e.g. STATE, BCL10), lymphocyte differentiation (P=3.5×10⁻³; e.g. CARD11), and regulation of apoptosis (P=1.9×10⁻³; e.g. BTG1, BTG2). Also significantly enriched were genes linked to transcriptional regulation (P=5.4×10⁻⁴; e.g. TP53) and genes involved in methylation (P=2.2×10⁻⁴) and acetylation (P=1.2×10⁻²), including histone methyltransferase (HMT) and acetyltransferase (HAT) enzymes known previously to be mutated in lymphoma (e.g. EZH2 [13] and CREBBP [15]).

Mutation hot spots can result from mutations at sites under strong selective pressure and such sites have previously been identified using RNA-seq data [13]. Therefore, RNA-seq data was searched for genes with mutation hot spots, and 10 genes were identified that were not mutated in the 14 genomes (PIM1, FOXO1, CCND3, TP53, IRF4, BTG2, CD79B, BCL7A, IKZF3 and B2M), of which five (FOXO1, CCND3, BTG2, IKZF3 and B2M) were not previously known targets of point mutation in NHL (Table 4). FOXO1, BCL7A and B2M exhibited hot spots affecting their start codons. The effect of a FOXO1 start codon mutation, which was observed in three cases, was further studied using a cell line in which the initiating ATG was mutated to TTG. Western blots probed with a FOXO1 antibody revealed a band with a reduced molecular weight, indicative of a FOXO1 N-terminal truncation (FIG. 4) consistent with utilization of the next in-frame ATG for translation initiation. A second hot spot in FOXO1 at T24 was mutated in two cases. T24 is reportedly phosphorylated by AKT subsequent to B-cell receptor (BCR) stimulation [16] inducing FOXO1 nuclear export.

The RNA-seq data was analysed to determine whether any of the somatic mutations in the 109 recurrently mutated genes showed evidence for allelic imbalance with expression favouring one allele. Of 380 expressed heterozygous mutant alleles, preferential expression of the mutation was observed for 16.8% (64/380) and preferential expression of the wild-type was observed for 27.8% (106/380). Seven genes displayed evidence for significant preferential expression of the mutant allele in at least two cases: (BCL2, CARD11, CD79B, EZH2, IRF4, MEF2B and TP53). In 27 of 43 cases with BCL2 cSNVs, expression favoured the mutant allele, consistent with the previously-described hypothesis that the translocated (and hence, transcriptionally deregulated) allele of BCL2 is targeted by somatic hypermutation [17]. Examples of mutations at known oncogenic hot spot sites such as F123I in CARD11 [18] exhibited allelic imbalance favouring the mutant allele in some cases. Similarly, expression favouring two novel hot spot mutations in MEF2B (Y69 and D83) was observed and two sites in EZH2 not previously reported as mutated in lymphoma (A682G and A692V).

To distinguish new cancer-related mutations from passenger mutations, the approach proposed by Greenman et al. was used [19]. 26 genes were identified with significant evidence for positive selection (FDR 0.03, Methods), with either selective pressure for acquiring non-synonymous point mutations or truncating/nonsense mutations (Table 1). Included were known lymphoma oncogenes (BCL2, CD79B [9], CARD11 [18], MYD88 [10] and EZH2 [13]), all of which exhibited signatures indicative of selection for non-synonymous variants.

Evidence for Selection of Inactivating Changes

Tumour suppressor genes were expected to exhibit strong selection for the acquisition of nonsense mutations. The eight most significant genes included seven with strong selective pressure for nonsense mutations, including the known tumour suppressor genes TP53 and TNFRSF14 [20] (Table 1). CREBBP, recently reported as commonly inactivated in DLBCL [15], also showed some evidence for acquisition of nonsense mutations and cSNVs (Table 5). Enrichment was observed for nonsense mutations in BCL10, a positive regulator of NF-κB, in which oncogenic truncated products have been described in lymphomas [21]. The remaining strongly significant genes (BTG1, GNA13, SGK1 and MLL2) had no reported role in lymphoma. GNA13 was affected by mutations in 22 cases including multiple nonsense mutations. GNA13 encodes the alpha subunit of a heterotrimeric G-protein coupled receptor responsible for modulating RhoA activity [22]. Some of the mutated residues negatively impact its function [23, 24], including a T203A mutation, which also exhibited allelic imbalance favouring the mutant allele. GNA13 protein was reduced or absent on Western blots in cell lines harbouring either a nonsense mutation, a stop codon deletion, a frame shifting deletion, or changes affecting splice sites (FIG. 5).

SGK1 encodes a PI3K-regulated kinase with functions including regulation of FOXO transcription factors [25], regulation of NF-κB by phosphorylating IkB kinase [26], and negative regulation of NOTCH signalling [27]. SGK1 also resides within a region of chromosome 6 commonly deleted in DLBCL (FIG. 1) [5]. The mechanism by which SGK1 and GNA13 inactivation may contribute to lymphoma is unclear but the strong degree of apparent selection towards their inactivation and their overall high mutation frequency (each mutated in 18 of 106 DLBCL cases) suggests that their loss contributes to B-cell NHL. Certain genes are known to be mutated more commonly in GCB DLBCLs (e.g. TP53 [28] and EZH2 [13]). Here, both SGK1 and GNA13 mutations were found only in GCB cases (P=1.93×10-3 and 2.28×10-4, Fisher exact test; n=15 and 18, respectively) (FIG. 2). Two additional genes (MEF2B and TNFRSF14) with no previously described role in DLBCL showed a similar restriction to GCB cases (FIG. 2).

Inactivating MLL2 Mutations

MLL2 exhibited the most significant evidence for selection and the largest number of nonsense SNVs was MLL2. RNA-seq analysis indicated that 26.0% (33/127) of cases carried at least one MLL2 cSNV. To address the possibility that variable RNA-seq coverage of MLL2 failed to capture some mutations, the entire MLL2 locus (˜36 kb) was PCR amplified in 89 cases (35 primary FLs, 17 DLBCL cell lines, and 37 DLBCLs). 58 of these cases were among the RNA-seq cohort. Illumina amplicon resequencing revealed 78 mutations, confirming the RNA-seq mutations in the overlapping cases and identifying 33 additional mutations. The somatic status of 46 variants was confirmed using Sanger sequencing (Table 6), and showed that 20 of the 33 additional mutations were insertions or deletions (indels). Three SNVs at splice sites were also detected, as were 10 new cSNVs that had not been detected by RNA-seq.

The somatic mutations were distributed across MLL2 (FIG. 3A). 37% (n=29/78) of these were nonsense mutations, 46% (n=36/78) were indels that altered the reading frame, 8% (n=6/78) were point mutations at splice sites and 9% (n=7/78) were non-synonymous amino acid substitutions (Table 2). Four of the somatic splice site mutations had effects on MLL2 transcript length and structure. For example, two heterozygous splice site mutations resulted in the use of a novel splice donor site and an intron retention event.

Approximately half of the NHL cases sequenced had two MLL2 mutations (Table 6). BAC clone sequencing was used in eight FL cases to show that in all eight cases the mutations were in trans, affecting both MLL2 alleles. This observation is consistent with the notion that there is a complete, or near-complete, loss of MLL2 in the tumour cells of such patients.

With the exception of two primary FL cases and two DLBCL cell lines (Pfeiffer and SU-DHL-9), the majority of MLL2 mutations appeared to be heterozygous. Analysis of Affymetrix 500 k SNP array data from two FL cases with apparent homozygous mutations revealed that both tumours exhibited copy number neutral loss of heterozygosity (LOH) for the region of chromosome 12 containing MLL2 (Methods). Thus, in addition to bi-allelic mutation, LOH is a second, albeit less common mechanism by which MLL2 function is lost.

MLL2 was the most frequently mutated gene in FL, and among the most frequently mutated genes in DLBCL (FIG. 2). MLL2 mutations were confirmed in 31 of 35 FL patients (89%), in 12 of 37 DLBCL patients (32%), in 10 of 17 DLBCL cell lines (59%) and in none of the eight normal centroblast samples sequenced. The analysis predicted that the majority of the somatic mutations observed in MLL2 were inactivating (91% disrupted the reading frame or were truncating point mutations), suggesting that MLL2 is a tumour suppressor of significance in NHL.

Recurrent Point Mutations in MEF2B

Selective pressure analysis also revealed genes with stronger pressure for acquisition of amino acid substitutions than for nonsense mutations. One such gene was MEF2B, which had not previously been linked to lymphoma. 20 (15.7%) cases had MEF2B cSNVs and 4 (3.1%) cases had MEF2C cSNVs. All cSNVs detected by RNA-seq affected either the MADS box or MEF2 domains. To determine the frequency and scope of MEF2B mutations, exons 2 and 3 were Sanger-sequences in 261 primary FL samples; 259 DLBCL primary tumours; 17 cell lines; 35 cases of assorted NHL (IBL, composite FL and PBMCL); and eight non-malignant centroblast samples. A capture strategy was also used to sequence the entire MEF2B coding region in the 261 FL samples, revealing six additional variants outside exons 2 and 3. 69 cases (34 DLBCL; 12.67% and 35 FL; 15.33%) were identified with MEF2B cSNVs or indels; novel variants in other NHL and non-malignant samples were not observed. 55 (80%) of the variants affected residues within the MADS box and MEF2 domains encoded by exons 2 and 3 (Table 7; FIG. 3B). Each patient generally had a single MEF2B variant and relatively few (8 total, 10.7%) truncation-inducing SNVs or indels were observed. Non-synonymous SNVs were by far the most common type of change observed, with 59.4% of detected variants affecting K4, Y69, N81 or D83. In 12 cases MEF2B mutations were shown to be somatic, including representative mutations at each of K4, Y69, N81 and D83 (Table 8). Mutations in ABC cases were not detected, indicating that somatic mutations in MEF2B play a role unique to the development of GCB DLBCL and FL (FIG. 2).

Discussion

In this study of genome, transcriptome and exome sequences from 127 B-cell NHL cases, 109 genes were identified with clear evidence of somatic mutation in multiple individuals. Significant selection appears to act on at least 26 of these for the acquisition of either nonsense or missense mutations. The majority of these genes do not appear to have previously been associated with any cancer type. An enrichment of somatic mutations was observed affecting genes involved in transcriptional regulation and, more specifically, chromatin modification.

MLL2 emerged from the analysis as a major tumour suppressor locus in NHL. It is one of six human H3K4-specific methyltransferases in the MLL family, all of which share homology with the Drosophila trithorax gene [29]. Trimethylated H3K4 (H3K4me3) is an epigenetic mark associated with the promoters of actively transcribed genes. By laying down this mark, MLLs are responsible for the transcriptional regulation of developmental genes including the homeobox (Hox) gene family [30] which collectively control segment specificity and cell fate in the developing embryo [31,32]. Each MLL family member is thought to target different subsets of Hox genes [33] and in addition, MLL2 is known to regulate the transcription of a diverse set of genes [34]. Recently, MLL2 mutations were reported in a small-cell lung cancer cell line [35] and in renal carcinoma [36] but the frequency of nonsense mutations affecting MLL2 in these cancers was not established in these reports. Parsons and colleagues recently reported inactivating mutations in MLL2 or MLL3 in 16% of medulloblastoma patients [37] further implicating MLL2 as a cancer gene.

The data in this example link MLL2 somatic mutations to B-cell NHL. The reported mutations are likely to be inactivating and in eight of the cases with multiple mutations, it was confirmed that both alleles were affected, presumably resulting in essentially complete loss of MLL2 function. The high prevalence of MLL2 mutations in FL (89%) equals the frequency of the t(14;18)(q32;q21) translocation, which is considered the most prevalent genetic abnormality in FL [3]. In DLBCL tumour samples and cell lines, MLL2 mutation frequencies were 32% and 59% respectively, also exceeding the prevalence of the most frequent cytogenetic abnormalities, such as the various translocations involving 3q27, which occur in 25-30% of DLBCLs and are enriched in ABC cases [38]. Importantly, MLL2 was found mutated in both DLBCL subtypes (FIG. 2). Analyses thus indicate that MLL2 acts as a central tumour suppressor in FL and both DLBCL subtypes.

The MEF2 gene family encodes four related transcription factors that recruit histone-modifying enzymes including histone deacetylases (HDACs) and HATs in a calcium-regulated manner. Although truncating variants were detected in MEF2 gene family members, the present analysis suggests that, in contrast to MLL2, MEF2 family members tend to selectively acquire non-synonymous amino acid substitutions. In the case of MEF2B, 59.4% of all the cSNVs were found at four sites within the protein (K4, Y69, N81 and D83), and all four of these sites were confirmed to be targets of somatic mutation. 39% of the MEF2B alterations affect D83, resulting in replacement of the charged aspartate with any of alanine, glycine or valine. Although the specific the consequences of these substitutions on protein function is unknown, it seems likely that their effect would impact the ability of MEF2B to facilitate gene expression and thus play a role in promoting the malignant transformation of germinal centre B cells to lymphoma.

MEF2B mutations can be linked to CREBBP and EP300 mutations, and to recurrent Y641 mutations in EZH2 [13]. One target of CREBBP/EP300 HAT activity is H3K27, which is methylated by EZH2 to repress transcription. There is evidence that the action of EZH2 antagonizes that of CREBBP/EP300 [39]. One function of MEF2 is to recruit either HDACs or CREBBP/EP300 to target genes [40], and it has been suggested that HDACs compete with CREBBP/EP300 for the same binding site on MEF2 [41]. Under normal Ca²⁺ levels, MEF2 is bound by type IIa HDACs, which maintain the tails of histone proteins in a deacetylated repressive chromatin state [42]. Increased cytoplasmic Ca²⁺ levels induce the nuclear export of HDACs, enabling the recruitment of HATs such as CREBBP/EP300, facilitating transcription at MEF2 target genes. Mutation of CREBBP, EP300 or MEF2B may impact expression of MEF2 target genes owing to reduced acetylation of nucleosomes near these genes (FIG. 6). In light of the recent finding that heterozygous EZH2 Y641 mutations enhance overall H3K27 trimethylation activity of PCR2 [43, 44], it is possible that mutation of both MLL2 and EZH2 could cooperate in reducing the expression of some of the same target genes. The data in this example show that (1) post-transcriptional modification of histones is of key importance in germinal centre B cells and (2) deregulated histone modification due to these mutations likely results in reduced acetylation and enhanced methylation and acts as a core driver event in the development of NHL (FIG. 6).

It is thought that GCB and ABC DLBCLs arise due to distinct genetic events [5] and it is widely accepted that the aggressive nature of the latter results from the acquisition of mutations that mimic stimulation of the B cell receptor by antigen or those that more directly induce constitutive activation of NF-κB [2]. This example provides other important modulators or components of BCR signalling and regulators of B cell differentiation or survival as targets of repeated and recurrent mutation, including MEF2B/C [79], SGK [5], IRF4 [82], STAT3 [77], STATE [83], RFTN1 [84], CCND3 [85], PLCG2, FOXO1 [86], CARD11 [18], CD79B [9] and MYD88 [10] and IKZF3 [87]. There were notable differences in mutation patterns among these genes. For example, MEF2B/C and STAT3, each of which function as dimers, showed strong evidence for selectively acquiring nonsynonymous (rather than truncating) mutations, whereas SGK1 and CCND3 appeared to be preferentially truncated in NHL. The previously characterized CARD11 [18], CD79B [9] and MYD88 [10] all act upstream of NF-κB, leading to its deregulation, typically in ABC DLBCLs. In the present Example, only CD79B and MYD88 (in addition to structural rearrangements involving BCL6) showed a significant enrichment for mutations in ABC cases (FIG. 2) and the point mutations observed largely corresponded to the known hot spots in these two genes [9, 10] (Table 4).

The remaining genes listed above encode proteins that are either activated or inhibited as a result of BCR stimulation, but not directly involved in regulating NF-κB. PRDM1 has been termed the plasma cell master differentiation gene as it orchestrates terminal differentiation of germinal centre B cells into plasma cells [88]. Importantly STAT3 [77], found here to be commonly mutated in DLBCL, regulates the activity or expression of PRDM1 in response to IL-21 stimulation. Of interest, inherited mutations in STAT3 are the primary cause of an immune disorder known as hyper IgE syndrome and it has been shown that in these cases mutant STAT3 acts in a dominant negative manner [89]. Strikingly, some of the somatic mutations reported here affect the same residues found mutated in the constitutional DNA of hyper IgE patients. This leads to a prediction that mutant cells may be unable to induce PRDM1 transcription following IL-21 stimulation (FIG. 6A). In particular, as many of these mutations were found in both GCB DLBCL and FL, the data suggests that malignant transformation of germinal centre B cells relies on components of BCR signalling separate from those utilized in ABC DLBCL (i.e. NF-κB) but also that altered regulation of PRDM1, previously thought to be a feature unique to ABC DLBCL, may be of general importance in NHL.

Mutations affecting CREBBP and EP300 were recently reported in DLBCL [15], and ALL [90]. Similar to the observations reported in these studies, the data shows a preference for accumulation of truncating SNVs (n=4, 16.7% of mutated cases) but also include non-synonymous SNVs in many cases (20 cases with cSNVs, Table 5). EP300 also contained multiple cSNVs (8 cases total). 3 EP300 cSNVs and 9 CREBBP cSNVs were confirmed as somatic mutations. Cases with multiple cSNVs in either gene were rarely observed (one cell line and three patients) consistent with the commonly held notion that both genes are haploinsufficient [91]. The cSNVs that were not predicted to result in protein truncation were mainly found within the HAT domain of these two proteins. These included four codons that are apparent mutation hot spots (Tables 4 and 5). Of these, three correspond to residues that have been reported to be homologous between the two proteins [75] (Table 5). Representative cSNVs corresponding to three of these hot spots in CREBBP and one in EP300 were confirmed as somatic. Three of the EP300 somatic non-synonymous mutations observed affected residues previously shown to reduce acetyltransferase activity in an in vitro acetyltransferase assay [75]. CREBBP (but not EP300) was confirmed to have a significant signature of selective pressure to acquire both truncating and missense mutations (Table 1), but the lack of significance for the latter may owe to limited statistical power due to its reduced mutation prevalence relative to CREBBP. Taken together, these data suggest that reduction or loss of either CREBBP or EP300 may promote lymphomagenesis. Of note, in contrast to a recent report [15], a significant difference was not observed in CREBBP or EP300 mutation frequency in the two subtypes (P=0.5656 for CREBBP and 0.6607 for EP300; Fisher exact test).

MEF2 proteins can act as transcriptional co-activators or co-repressors by recruiting two classes of enzymes that alter the acetylation state of histone tails, namely HATs and HDACs. MEF2 dimers are known to associate with the two HATs CREBBP and EP300 [30] and it has been suggested that HDACs and CREBBP/EP300 compete for the same binding site on MEF2 [41]. Under normal levels of intracellular Ca²⁺, MEF2 is bound by one of several type IIa HDACs, which maintain the tails of histone proteins in a deacetylated repressive chromatin state [42]. Increased cytoplasmic Ca²⁺ levels induce the nuclear export of the bound HDAC, thus enabling MEF2 dimers to recruit a HAT enzyme such as CREBBP/EP300, which facilitate transcription at MEF2 target genes by catalysing the addition of acetyl groups to the tails of core histone proteins including lysine 27 on histone H3 (H3K27) [40, 41] (FIG. 6D).

Ca²⁺-mediated induction of MEF2 target genes is utilised in diverse developmental processes including muscle and neuronal cell differentiation [92] as well as the maturation of B and T cells [80]. For example, during negative selection, upon T-cell-receptor (TCR) stimulation, the resulting Ca²⁺ influx results in MEF2-mediated induction of the pro-apoptosis NR4A1 (NUR77), which, in turn drives apoptosis of self-reactive T cells [80]. It has also been shown in T cells that MEF2D interacts directly with nuclear NFAT, another Ca²⁺/CaM-regulated protein, and recruits EP300 to MEF2 target genes [93]. In murine B cells, it was recently demonstrated that MEF2C is required to mediate gene expression events following BCR stimulation, but this study did not discuss a possible overlapping role of MEF2B in this process nor was there a conclusive identification of the MEF2C-regulated genes important to this process [79]. That mutations in MEF2C were also observed at a lower frequency in NHL samples supports the interpretation that these proteins share a related function in this cellular context. The MEF2B dimer has previously been co-crystallized with three of its interacting partners, namely Cabin1 [81], HDAC9 [41] and EP300 [50] and, informed by these structures, one could predict that many of the recurrent mutations would negatively impact the function of MEF2B. For example, at least three of the mutated residues (K5, K23 and R24) are required for mediating the binding of MEF2 to DNA [94]. Because MEF2 proteins can heterodimerize [95], mutations that impact the function of MEF2 are known to produce a dominant effect on the overall function of any MEF2-family protein by occupying a significant proportion of MEF2-containing complexes [96]. In fact one of the residues found mutated in this study (K24) was previously demonstrated to act as a dominant negative when ectopically expressed [96]. Further, the mutation hot spot Y69 was recently shown to be involved in multiple interactions in a solved crystal structure of MEF2B bound to EP300 [50], suggesting the possibility that this mutation may impact the ability of these two proteins to interact. Although the impact of the individual MEF2B mutations on MEF2 function requires further study, the recurrence of these mutations among a limited set of residues suggests the action of positive selection for these mutations during cancer progression.

When one considers the high frequency of mutations detected that affect genes encoding MEF2 proteins, it is striking that inactivating mutations affecting both CREBBP and EP300 are common in NHL, as these are both known effectors of the induction of MEF2-regulated genes. Notably, with one exception, all of the truncation-inducing mutations identified in CREBBP and EP300 are predicted to remove the histone acetyltransferase (HAT) domain of the protein [81]. Moreover, comparison of the positions mutated in CREBBP to those mutated in EP300 reveals that some homologous residues within the HAT domains are affected in both proteins. Based on the crystal structure of EP300, five of these recurrently mutated residues were previously identified as important for mediating substrate interaction [75]. In that study, three of these residues were mutated and showed loss (or reduction) of HAT activity in vitro, suggesting that many of the cSNVs observed in these two proteins also negatively impact their function in vivo. Further, CREBBP/EP300 are both known to regulate the function of FOXO1 [97], another gene found recurrently mutated in this study. Thus it is also possible that the mutation of these genes in addition their potential effect on MEF2-mediated transactivation, could impact the normal AKT-mediated nuclear exclusion of FOXO1 (FIG. 6C).

The data presented herein is consistent with a model wherein the induction of MEF2 target genes in response to BCR stimulation is inhibited by mutations that reduce the function of MEF2 complexes, potentially in a dominant negative fashion, or mutations that inactivate either of their transcriptional co-activators CREBBP or EP300 (FIG. 6D). Another mutation identified herein in a single case is also consistent with this model, namely the mutation of S155 to phenylalanine in HDAC7. This serine residue is known to be phosphorylated by CAMK following TCR stimulation, facilitating nuclear export of HDAC7 in response to Ca²⁺ influx [98]. In the cited study, mutation of this residue resulted in impaired export of HDAC7 following TCR stimulation thereby inhibiting MEF2-mediated induction of NUR77 expression and hence, inhibiting NUR77-mediated apoptosis. Thus, this mutant could potentially produce a nuclear-restricted protein that leads to constitutive suppression of MEF2 target genes regardless of intracellular Ca²⁺ levels. This would be a similar effect that would be expected for loss-of-function mutations of MEF2B, CREBBP or EP300. Though an increase in cytoplasmic Ca²⁺ is one downstream signal following BCR stimulation, the NFAT transcription factors, key downstream mediators of this signal that promote survival, were not mutated and thus are expected to function normally. Also, pathways such as NF-κB and events modulated by AKT do not rely on the Ca²⁺ messenger and should therefore be unaffected by these mutations. Interestingly, a recent report suggests that SGK1 (found here to be commonly inactivated in DLBCL) may also play a role in modulating Ca²⁺ levels by regulating the turnover of the Ca²⁺ channel protein Orai [99]. Thus, this model predicts that mutations directly affecting MEF2 function (i.e. those in MEF2B, MEF2C, HDAC7, CREBBP or EP300) or other genes involved in regulating cytoplasmic calcium levels would diminish the cell's ability to induce MEF2 target genes in response to BCR stimulation while leaving other downstream signals intact.

Methods

Sample Acquisition

Lymphoma samples were classified by an expert haematopathologist (R.D.G) according to the World Health Organization criteria of 2008. Benign specimens included reactive pediatric tonsils or purified CD77-positive centroblasts sorted from reactive tonsils using Miltenyi magnetic beads (Miltenyi Biotec, CA). The tumour specimens were collected as part of a research project approved by the University of British Columbia-British Columbia Cancer Agency Research Ethics Board (BCCA REB) and are in accordance with the Declaration of Helsinki.

For all DLBCL samples profiled by RNA-seq, genome or exome sequencing in this study, tumour content was greater than 50% as assessed by: a) immunophenotyping using flow cytometry to detect the level of coexpression of CD19 and light chain restriction; or b) a pathologist review of an H&E-stained frozen section taken adjacent to the tissue that was cut and used for nucleic acid extraction. All other specimens used in this study were obtained at the time of diagnosis and were derived from archived fresh-frozen tissue or frozen tumour cell suspensions. Constitutional DNA was obtained from peripheral blood or from B cell-negative sorted tumour cell suspensions (fraction eluted from cells captured by B Cell Isolation Kit II or CD19 MicroBeads (Miltenyi Biotec, CA)).

Cell Lines

DB [51], DOHH-2 [52], Karpas422 [53], NU-DHL-1 [54], NU-DUL-1 [55], SU-DHL-6 and WSU-DLCL2 [56] are cell lines obtained from DSMZ. Pfeiffer and Toledo were obtained from ATCC and all OCI-Ly [57] lines (1, 3, 7, 10 and 19) were obtained from Louis Staudt (US National Institutes of Health). The cell lines MD903, SU-DHL-9 and RIVA were obtained from Martin Dyer (University of Leicester, UK).

Preparation and Sequencing of RNA-Seq, Genome and Exon Capture Illumina Libraries

Genomic DNA for construction of genome and exome libraries was prepared from biopsy materials using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen). DNA quality was assessed by spectrophotometry (260 nm/280 nm and 260 nm/230 nm absorption ratios) and gel electrophoresis before library construction. DNA was sheared for 10 minutes using a Sonic Dismembrator 550 with a power setting of “7” in pulses of 30 seconds interspersed with 30 seconds of cooling (Cup Horn, Fisher Scientific) and then analysed on 8% PAGE gels. The 200 to 300 bp DNA size fraction was excised and eluted from the gel slice overnight at 4° C. in 300 μL of elution buffer (5:1 (vol/vol) LoTE buffer (3 mM Tris-HCl, pH 7.5, 0.2 mM EDTA)/7.5 M ammonium acetate) and was purified using a Spin-X Filter Tube (Fisher Scientific) and ethanol precipitation. Genome libraries were prepared using a modified paired-end protocol supplied by Illumina Inc. This involved DNA end-repair and formation of 3′ adenosine overhangs using the Klenow fragment of DNA polymerase I (3′-5′ exonuclease minus) and ligation to Illumina PE adapters (with 5′ overhangs). Adapter-ligated products were purified on QIAQUICK® spin columns (Qiagen) and PCR-amplified using PHUSION® DNA polymerase (NEB) and ten cycles with the PE primer 1.0 and 2.0 (Illumina). PCR products of the desired size range were purified from adapter ligation artifacts using 8% PAGE gels. DNA quality was assessed and quantified using an Agilent DNA 1000 series II assay (Agilent) and Nanodrop 7500 spectrophotometer (Nanodrop), and DNA was subsequently diluted to 10 nM. The final concentration was confirmed using a QUANT-IT™ dsDNA HS assay kit and Qubit fluorometer (Invitrogen).

For genomic DNA sequencing, clusters were generated on the Illumina cluster stations using v1 cluster reagents. Paired-end reads were generated using v3 sequencing reagents on the Illumina GA_(iix), platform following the manufacturer's instructions. Image analysis, base-calling and error calibration were performed using v1.0 of Illumina's Genome analysis pipeline. The DLBCL genomes were sequenced with 100 nucleotide paired-end reads using the HiSeq2000 platform. For RNA-seq analysis, a modified method was used similar to the protocol previously described [13]. Briefly, RNA was extracted from 15×20 μm sections cut from fresh-frozen lymph node biopsies using the MACS mRNA isolation kit (Miltenyi Biotec), from 5-10 μg of DNase I-treated total RNA as per the manufacturer's instructions. Double-stranded cDNA was synthesized from the purified poly(A)⁺RNA using the SUPERSCRIPT® Double-Stranded cDNA Synthesis kit (Invitrogen) and random hexamer primers (Invitrogen) at a concentration of 5 μM. The cDNA was fragmented by sonication and a paired-end sequencing library prepared following the Illumina paired-end library preparation protocol (Illumina).

For exome sequencing, genomic DNA was extracted following the protocol supplied in the Qiagen AllPrep DNA/RNA Mini Kit (Cat#80204), and quantified using a QUANT-IT™ dsDNA HS assay kit and a Qubit fluorometer (Invitrogen). Approximately 500ng DNA was sheared for 75 seconds at duty cycle “20%” and intensity of “5” using a Covaris E210, and run on an 8% PAGE gel. A 200 to 250 bp DNA size fraction was excised and eluted from the gel slice, and was ligated to Illumina paired-end adapters following a standard protocol as previously described [13]. The adapter ligated DNA was amplified for 10 cycles using the PE primer set (Illumina) and purified as a pre-exome capture library. The DNA was assessed using an Agilent DNA 1000 Series II assay, and 500ng DNA was hybridized to the 38 Mb Human exon probe using the All Exon Kit (Cat#G3362) following the Agilent SureSelect Paired-End Target Enrichment System Protocol (Version 1.0, September 2009). The captured DNA was purified using a Qiagen MinElute column, and amplified for 12 cycles using PE primer set. The PCR products were run on an 8% PAGE gel, the desired size range (320 to 370 bp) was excised and purified, and was then assessed using an Agilent DNA 1000 series II assay and diluted to 10 nM. The final library DNA concentration was confirmed using a QUANT-IT™ dsDNA HS assay kit and Qubit fluorometer. Clusters were generated on the Illumina cluster station and paired-end reads generated using an Illumina Genome Analyzer (GA_(IIx)) following the manufacturer's instructions.

Alignment-Based Analysis of Tumour DNA and RNA Sequence for Somatic Point Mutations

All reads were aligned to the human reference genome (hg18) or (for RNA-seq) to a genome file that was augmented with a set of all exon-exon junction sequences using BWA version 0.5.4 [46]. RNA-seq libraries were aligned with an in-house modified version of BWA that is aware of exon junction reads and considers them when determining pairing distance in the “sampe” (read pairing) phase of alignment. Candidate single-nucleotide variants (SNVs) were identified in the aligned genomic sequence reads and the transcriptome (RNA-seq) reads using an approach similar to one we previously described [13]. One key difference in the variant calling in this study was the application of a Bayesian SNV identification algorithm (‘SNVmix’) [47]. This approach is able to identify SNVs with a minimum coverage of two high-quality (Q20) bases. SNVs were retained if they had a SNVmix probability of at least 0.99 and had support from reads mapping to both genomic strands. Any SNV near gapped alignments or exactly overlapping sites assessed as being polymorphisms (SNPs) were disregarded, including variants matching a position in dbSNP or the sequenced personal genomes of Venter [58], Watson[59] or the anonymous Asian[60] and Yoruban[61] individuals. For paired samples with matched constitutional DNA sequence, all variants with evidence (a SNVmix probability of at least 0.99 and 2 or more high quality base calls matching the SNV) in the constitutional DNA were considered germline variants and were no longer considered cSNVs.

Mutations were annotated on genes using the Ensembl transcripts (version 54), except in the cases of MEF2B and MLL2, for which the Ensembl annotations were deemed inferior to the Refseq. Because situations were observed where exons were represented in Ensembl transcripts that were not also represented in a Refseq, candidate mutations are only reported in exons shared by both annotations (e.g. in Supplementary Table S4). Candidate mutations were subsequently reviewed visually in the integrative genomics viewer (IGV) [62] and those appearing to be artefacts or with some evidence (2 or more reads) visible in the constitutional DNA sequence were removed.

Validation of Candidate Somatic Mutations Using Illumina Sequencing

Validation was accomplished by designing primers to amplify a 200 to 300 by region around the targeted variant with one primer within reach of a single read (i.e. maintaining the sum of the primer length and distance to variant less than 100 bp, depending on read length used). Amplicons were generated for both tumour and normal DNA. Two pools of amplicons were generated, one for tumour and one for normal DNA, with equal volumes from each PCR reaction (or increased volume for amplicons that resulted in faint bands in an agarose gel) and an Illumina paired-end sequencing library was constructed from the pool. For variants common to more than one patient, a 6nt index, which was added to the 5′ end of each primer, was assigned for each patient. These index sequences were trimmed from sequence reads prior to alignment and subsequently used to associate the data with individual patients. Reads were aligned using BWA and variants were visually confirmed for validity and somatic status in IGV [63] (absence from constitutional DNA). Variants with primer design or PCR failures were scored as ‘unvalidated’.

Validation of cSNVs by Sanger Sequencing

The majority of candidate cSNVs were validated by Sanger sequencing of the region surrounding each mutation. These included all cSNVs identified in the two DLBCL exomes and the FL genome/exome (i.e. DLBCL-PatientA, DLBCL-PatientB and FL-PatientA). For the additional DLBCL genomes, cSNVs were selected for validation only if there were three or more cSNVs in that gene in the entire cohort. To do so, primers were designed to amplify 350-1200 bp regions by PCR (most amplicons were ˜400 bp). Forward and reverse primers were tailed with T7 and M13Reverse 5′ priming sites, respectively. PCR conditions used were 94° C. for 2 minutes, 30 cycles of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute, and a final extension at 72° C. for 8 minutes. To determine the somatic or germ line origin of the mutations, mutations were re-sequenced in both tumour and constitutional DNA, the latter obtained from peripheral blood or negative-sort cells (see section entitled Sample Acquisition). The sequencing reactions consisted of 50 cycles of 96° C. for 10 seconds, 43° C. (for M13Reverse) or 48° C. (T7) for 5 seconds and 60° C. for 4 minutes and were analysed using an AB 3730XL. All capillary traces were analysed using the Staden Package [64] and all somatic variants were visually inspected to confirm their presence in tumour and absence from germ line traces. Some regions that failed to amplify in the first attempt were re-addressed with the addition of 5% DMSO and 5% betaine to the sequencing reactions, but otherwise maintaining the PCR conditions. SNVs in certain genes, such as BCL7A and HDAC7, repeatedly failed to amplify and for these, it was not possible to address whether the mutations in these genes were somatically acquired or were present in the germ line. Validation was not performed for variants in BCL2 or CD79B as their somatic mutation status in DLBCL is well established.

Detection of Enrichment of Functional Gene Classes within Frequently Mutated Genes

Significant functional classes represented in the cSNV list were identified using the DAVID Functional Annotation tool. Reported P values were corrected for multiple testing using the Benjamini method.

Detection of Mutations with Imbalanced/Skewed Expression

The analysis of imbalanced expression was restricted to (1) confirmed somatic nonsynonymous point mutations along with (2) previously published hot spot mutations. In total, there were 381 such mutations in 99 of the 109 genes represented in the RNA-seq data. For each mutated gene, the number of aligned reads supporting the reference and mutant allele was determined. For genes with multiple mutations in the same patient (e.g. BCL2), the sum of all reads supporting each of the non-reference alleles in that patient was used instead (assuming that all mutations were restricted to the same allele). Significant imbalance/skew was computed using the binomial exact test and P values were corrected using the Bonferroni method.

Calculation of Selective Pressure

To determine if mutational patterns were indicative of selective pressure, both synonymous and non-synonymous cSNVs were considered across the patient cohort (excluding those found to be present in the germ line or false positives after validation). Selection can be inferred when the type of mutations in a gene differs from those expected by chance given a specific mutation profile. To analyse the significance of this deviation, methods described by Greenman and colleagues [20] were applied to identify genes with signatures of selection. This analysis was performed on the 101 (of 109 total) genes that had, in addition to 2 or more confirmed somatic mutations, more than 2 cSNVs in total. The coding sequence of each gene (using the longest Refseq annotation for that gene) was scanned for all possible silent and non-silent mutations (missense and truncating) matching six types of sequence changes (C>A, C>G, C>T, T>A, T>C, T>G). The separation of mutations into different strata allows the model to consider the overall effect that cancer specific mutation mechanisms may have on the mutation profile. A null-selection mutation profile is estimated via the synonymous mutations, under the assumption that they do not confer an advantage to the tumour. A score statistic describing the selective pressure was then calculated by comparing the expected mutations of each type to the observed ones. Statistical significance was then determined by constructing an empirical distribution of scores from 100,000 Monte Carlo simulations under the null hypothesis of no selection. The number of Monte Carlo iterations was increased to a maximum of 14,600,000 for genes that did not obtain a p-value at the default 100,000 simulations. The type and strength of the selective pressure the genes were under were also estimated using the models described by Greenman et al. [20]. This is represented by a quantitative value of less than, equal to, or larger than 1 for negative, null, or positive selection respectively (Table 1, other data not shown).

Several genes in the list have previously been identified as targets of somatic hypermutation (SHM), which is mediated by the enzyme AICDA (also known as AID) and targets a limited number of genes in DLBCL [65, 66]. In an attempt to avoid biasing the selective pressure model with the distinct mutational signature caused by somatic hypermutation, the genes were split into two sets. The hypermutation set contained genes previously reported to be targets of SHM (BCL2 [17], BCL6, IRF4, PIM1, and CIITA) and the non-hypermutation set contained the remaining 95 genes. The effect of the different mutational profiles of both sets can be appreciated by considering the BCL2 case. When inserted into the model with the rest of the genes BCL2 presented the highest selective pressure of all genes (65.65); however, when the selective pressure model was applied to the hypermutated genes separately, BCL2 selective pressure was estimated at 3.78.

Identifying Genes with Mutation Hot Spots

Hot spots were identified by searching for clustered mutations in the cSNVs identified by RNA-seq. Owing to the lack of constitutional DNA sequence from some patient samples, whether the variants detected only by RNA-seq were present in the germ line could not necessarily be discerned. Cases were sought in which codons were recurrently mutated. To find hot spots in the RNA-seq data, a search was performed for sets of distinct variants producing non-synonymous changes affecting the same codon in different tumours. The genes that met this criterion (Table 4) included known targets of recurrent mutation (EZH2, CARD11 [18] and CD79B [9]) and three hot spots in MEF2B. Also among these genes were known targets of aberrant somatic hypermutation in DLBCL, including BCL2, IRF4 [65], PIM1 [66], BCL6 [67], and BCL7A [65].

Analysis of Aligned Genomic DNA Sequence for Copy Number Alterations and LOH

For the identification of copy number variations (CNVs), sequence quality filtering was used to remove all reads of low mapping quality (Q≤10). Due to the varying numbers of sequence reads from each sample, aligned reference reads were first used to define genomic bins of equal reference coverage to which depths of alignments of sequence from each of the tumour samples were compared. This resulted in a measurement of the relative number of aligned reads from the tumours and reference in bins of variable length along the genome, where bin width is inversely proportional to the number of mapped reference reads. After an estimate of differential GC bias was used to reduce noise, an HMM was used to classify and segment continuous regions of copy number loss, neutrality, or gain using methodology outlined previously [68].

Loss of heterozygosity was determined for each sample using the lists of genomic SNPs that were identified through the BWA/SNVMix pipeline. This analysis allows for classification of each SNP as either heterozygous or homozygous based on the reported SNP probabilities. For each sample, genomic bins of consistent SNP coverage were used by an HMM to identify genomic regions of consistent rates of heterozygosity. The HMM partitioned each tumour genome into three states: normal heterozygosity, increased homozygosity (low), and total homozygosity (high). It can be inferred that a region of low homozygosity either represents a state where only a portion of the cellular population had lost a copy of a chromosomal region or the signal was convoluted due to contaminating normal cells in the tumour. Both states of reduced homozygosity are displayed in blue in FIG. 1, generated by Circos [69].

Assembly-Based Analysis of Tumour DNA and RNA Sequence

Reads from the individual RNA-seq libraries were assembled using ABySS as previously described [70] using multiple values of k. Iterative pairwise alignments of the contigs from the individual kmer assemblies resulted in a merged contig set that was aligned against the reference Human genome (hg18) using BLAT as described [48]. Putative fusions were identified from contigs that had alignments to two distinct genomic locations. The putative events were filtered using evidence from alignment of reads to contigs using Bowtie and alignments of reads to the genome using BWA. Those events with at least four read pairs from the reads-to-genome alignment and two supporting reads from the reads-to-contig alignment (i.e. across the fusion breakpoint) were manually curated to produce a final list of putative fusions. The genomic breakpoints for the transcriptome predicted events were identified manually from the alignments of the reads to the genome using IGV. The genomic breakpoints were later confirmed by assembly using ABySS.

Putative indels were identified from alignment of the contigs to hg18 using BLAT when contiguous unmatched base(s) were found in either the contig (insertion) or reference (deletion) sequences. The events were filtered for read support with events requiring three or more reads to be considered in the filtered set. The filtered set was then screened against dbSNP130 to find putative novel events. The resulting set was manually inspected using read alignments (against both the genome and contigs) to visually confirm candidates. This approach revealed the deletion in GNA13 shown in FIG. 5.

The splicing alterations in MLL2 (FIGS. 3B and C) and GNA13 (FIG. 5) were identified from pairwise alignments of the contigs to hg18 using BLAT. The contig alignments were then matched against the four known gene models to identify novel splice junctions. The putative novel splice junctions were filtered where two or more reads were required across the novel junction for the event to be considered. Manual inspection using read alignments (against both the genome and contigs) was performed to visually confirm candidates.

Cell of Origin Subtype Assignment Using RNA-Seq Expression Values

Global gene expression signatures measured with microarrays are the standard method for classifying DLBCL samples into the two molecular subtypes (GCB and ABC). The Bayesian method described by Wright et al. [50] was adapted to allow classification to be accomplished with the expression values obtained from RNA-seq data. To accomplish this, expression values for each Ensembl gene model (version 54) were computed as FPKM (fragments per kilobase gene model per million, rather than RPKM to account for the use of paired-end reads) and log-transformed. The current standard approach for routinely classifying samples using Affymetrix U133 arrays employs 186 probesets (George Wright, personal communication). The 165 Ensembl genes that correspond to these probesets were used for classification by RNA-seq. The classifier was trained using the 43 cases previously classified as GCB and 21 classified as ABC using Affymetrix data. The FPKM values for these genes were compared between the samples with known subtypes using the T test and those producing a P value <0.01 were used for the classifier. The robustness of this approach was tested using leave-one-out cross-validation, which resulted in no mis-classifications. Similarly, no samples were mis-classified when all cases with known COO (based on Affymetrix data) were used to produce the classifier however there were some cases that were defined as unclassifiable (U) by one method and given a subtype assignment by the other method. In such cases, the subtype assignment (rather than U) was used.

Targeted MEF2B Resequencing Using Biotinylated RNA Capture Probes

The following strategy was used to sequence the entire MEF2B locus in multiple patient samples in multiplex. Four exonic regions of the MEF2B gene were amplified from a template consisting of a pool of DNAs from three bacterial artificial chromosomes (BACs) containing the MEF2B locus (M. Nefedov, P. J. de Jong and U Surtiby, unpublished) using PCR. PCR reactions consisting of 0.5 Units PHUSION® DNA Polymerase (New England Biolabs, Pickering, Ont.), 0.25 mM dNTPs, 3% DMSO, 0.4 μM of the forward and reverse primer and 5 pmol template were cycled on a MJR Pelletier Thermocycler (model PTC-225) for 30 seconds at 980° C.; 25×{10 seconds at 98° C., 30 seconds at 65° C., 30 seconds at 72° C.}; 5 minutes at 720° C. The resulting PCR amplicons, ranging in size from 342 to 474 bp, were size selected on an 8% NOVEX®-TBE gel (Invitrogen Canada Inc., Burlington, Ont.), excised and eluted into 300 μL of elution buffer containing 5:1 (vol/vol) LoTe (3 mM Tris-HCl, pH7.5, 0.2 nM EDTA)/7.5 M ammonium acetate. The eluates were purified from gel slurries by centrifugation through Spin-X centrifuge tube filters (Fisher Scientific Ltd., Nepean, Ont.), and EtOH precipitated. Purified amplicon DNAs were quantified using an Agilent DNA 1000 Series II assay (Agilent Technologies Canada Inc., Mississauga, Ont.). Individual amplicons were pooled (equimolar) and sheared using the Covaris S2 focused ultra-sonicator (Covaris Inc., Woburn, Mass.) with the following settings; 10% Duty cycle, 5% Intensity, and 200 Cycles per burst for 180 seconds. The resulting products were size fractioned on an 8% NOVEX® TBE gel (Invitrogen Canada Inc.) and the 75 to 125 bp fraction isolated, purified and quantified as above. 30 ng of resulting DNA was end-repaired, 3-prime modified with Adenosine overhangs, and ligated to custom adapters containing T7 and T3 promoter sequences as described [71]. Adapter-ligated products were enriched by PCR as above using T3 and T7 sense strand-specific primers and the following cycling conditions; 1 min. at 980C; 8×{10 seconds at 98° C., 30 seconds at 60° C., 30 seconds at 72° C.}; 5 minutes at 720° C. The amplified products were separated from excess adapter on an 8% NOVEX® TBE gel (Invitrogen Canada Inc.), purified, and quantified using the Quant iT™ QUANT-IT′ assay and Qubit Fluorometer (Invitrogen Canada Inc.). An in vitro transcription reaction was carried out using 100 ng of purified adapter-ligated DNA as per the manufacturer's specifications (AMPLISCRIBE™ T7-FLASH™ Biotin-RNA Transcription Kit; Intersciences Inc., Markham, Ont.). The reaction mixture was incubated at 37° C. for 60 minutes, DNase-I treated for 15 minutes at 37° C., and then incubated at 70° C. for 5 minutes to inactivate DNasel. Transcription products were precipitated with 1 volume of 5M NH4Ac, and size fractioned on a 10% NOVEX® TBE-Urea gel (Invitrogen Canada Inc.). The 100 to 150 bp fraction was isolated from the gel, eluted into 0.3M NaCl, and EtOH-precipitated after extraction of the eluate from the gel slurry by centrifugation through a Spin-X Filter centrifuge tube filter (Fisher Scientific Ltd.). The biotinylated RNA was resuspended in 20 μl nuclease-free water and quantified using an Agilent RNA Nano assay (Agilent Technologies Canada Inc.).

Indexed libraries of patient genomic DNA were pooled from 96 well plates in groups ranging from 36 to 47 libraries per pool [72]. A 250 to 350 bp size fraction from each pool was size-selected by gel purification from an 8% NOVEX® TBE gel as above (Invitrogen Canada Inc.). The protocol described by Gnirke and colleagues [73] was followed for the hybridization reaction and subsequent washes, with an additional oligonucleotide block consisting of standard Illumina PCR primers PE1 and PE2 included in the hybridization reaction mixture to prevent cross-hybridization between library fragments. The incubation of the library fragments with the RNA probe pool was carried out for 24 hours at 65° C., followed by binding to M-280 Streptavidin DYNABEADS® (Invitrogen Canada Inc.), washes, and elution of the captured library fragments. The eluted fragments were amplified by PCR using primers that anneal upstream of the adapter index sites and subjected to cluster generation and sequencing as described above.

Targeted MLL2 Resequencing Using Long-Range PCR and Sample Indexing

Due to the presence of inactivating mutations in different positions within the MLL2 gene, the entire MLL2 locus (chr12:47,699,025-47,735,374; hg18) was sequenced in a cohort of 35 FL and 37 DLBCL primary tumours, in 17 DLBCL derived cell lines and, as a control, in 8 centroblast samples. Genomic DNA from individual samples was normalized to 5 ng/μl, and 12.5 ng of each sample was PCR amplified using LA Taq DNA polymerase (TaKaRa). Twelve long amplicons, of sizes ranging from 6600 bp to 7800 bp, were obtained under the following PCR conditions: 94° C. for 5 minutes, 35 cycles of 98° C. for 10 seconds and 68° C. for 8 minutes, and a final extension at 72° C. for 10 minutes. Amplicons were cleaned using AMPure beads (Beckman Coulter) and eluted with 20-μL of TE. All 12 amplicons per sample were normalized and pooled together.

An individual indexed library was constructed from each sample (comprising the pool of the 12 long amplicons from MLL2). Approximately 500 ng of each pooled DNA sample was sheared for 10 min using a Sonic Dismembrator 550 with a power setting of “7” in pulses of 30 seconds interspersed with 30 seconds of cooling (Cup Horn, Fisher Scientific) and then analysed on 8% PAGE gels. The 200 to 300 bp DNA fraction was excised and eluted from the gel slice overnight at 4° C. in 300 μL of elution buffer (5:1 (vol/vol) LoTE buffer (3 mM Tris-HCl, pH 7.5, 0.2 mM EDTA)/7.5 M ammonium acetate) and was purified using a Spin-X Filter Tube (Fisher Scientific) and by ethanol precipitation. Indexed libraries were prepared using a modified paired-end protocol. This involved DNA end-repair reactions at room temperature 20-25° C. for 30 minutes (5 U T4 DNA polymerase, 1 U Klenow DNA polymerase (exonuclease minus), 100 U T4 polynucleotide kinase and 0.4 mM dNTP mix (Invitrogen). End-repair reactions were purified using AMPure beads, and dATP was added to the 3′ ends using 5 U Klenow DNA polymerase (exonuclease minus) and 0.2 mM dATP in 1× Klenow Buffer (Invitrogen) with 30-minute incubation at 37° C. in a Tetrad thermal cycler (MJ Research). DNA was again purified on AMPure beads using a Biomek FX. Adapter ligation (10:1 ratio) was completed with 0.03 μM adapter (multiplexing adapters 1 and 2), 100 ng DNA, 5 U T4 DNA ligase, 0.2 mM ATP and 1×T4 DNA Ligase Buffer (Invitrogen) for 30 minutes at room temperature. Adapter-ligated DNA was again purified using AMPure beads on a Biomek FX. A selection of DNA samples were quantified on a Qubit (Invitrogen). 15-cycle indexing enrichment PCR was performed using Phusion DNA polymerase and Primers 1.0 and 2.0 (IDT) and 96 custom indexing primers. PCR cycles were: 98° C. for 60 seconds, followed by 15 cycles of 98° C. for 10 seconds, 65° C. for 15 seconds and 72° C. for 30 seconds. The PCR products were purified using AMPure beads and eluted in 40 μL elution buffer EB (Qiagen). Product quality was assessed by quality-control gels with 1.75% SeaKem LE agarose in 1×TAE (0.2 μL of every amplicon) and on a 2100 Bioanalyzer (Agilent Technologies).

Indexed libraries were pooled together and sequenced on two lanes of a flowcell using an Illumina GA_(II) platform. Individual indexes allowed the deconvolution of reads deriving from individual samples in multiplexed libraries such that many cases were concurrently sequenced in the same flow cell lane. The reads were matched to patient samples using the index read and were aligned with BWA to the human reference genome (hg18). Point mutations were identified using SNVMix with stringent post-filtration including a requirement for dual-strand coverage and requiring at least 10% of the aligned reads at a candidate variant to be non-reference. Insertions and deletions were identified using the SAMtools indel calling algorithm with similar filters. Only insertions and deletions supported by at least 2 reads on each strand were considered valid. The reported average coverage for each sample was calculated as the average depth of aligned reads across each of the coding (CDS) positions in the MLL2 locus.

Re-Confirmation of MLL2 Mutations in Patient Samples and DLBCL Cell Lines

MLL2 mutations found by targeted sequencing of MLL2 in lymphoma samples were validated by Sanger sequencing of the region surrounding each mutation, except in 15 cases. To do so, primers were designed to amplify 400-600 by regions by PCR. Validating forward and reverse primers carried T7 and M13Reverse 5′ tails, respectively. PCR conditions used were 94° C. for 2 minutes, 30 cycles of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute, and a final extension at 72° C. for 8 minutes. To determine the somatic or germline origin of the mutations, mutations were re-sequenced in both tumour and constitutional DNA, the latter obtained from peripheral blood or negative sort cells. The sequencing reactions consisted of 50 cycles of 96° C. for 10 sec, 43° C. (for M13Reverse) or 48° C. (T7) for 5 seconds and 60° C. for 4 minutes and were analysed using an AB 3730XL. Variants were visually inspected to confirm their presence in tumour and absence from germline traces. In 8 of the patient samples that carried 2 mutations in MLL2, to establish whether one allele contained both mutations or each allele contained one, we sequenced both candidate mutations using DNA from BAC clones from FL patient libraries. The primers and PCR conditions were the same as those used for the validation of each of those mutations.

Targeted Resequencing of MEF2B Coding Exons 1 and 2

Coding exons 1 and 2 of MEF2B were PCR amplified using MEF2B_1F/R and MEF2B_2F/R primers using the same conditions for MLL2 (previous paragraph). Priming sites for T7 and M13Reverse were added to their 5′ ends to allow direct Sanger sequencing of amplicons. Amplicons were produced from whole genome amplified tumour genomic DNA from lymphoma patients and DLBCL cell lines. Whole genome amplification was performed using REPLI-G® Screening kit reagents (Qiagen), following the manufacturer instructions. All capillary traces were visually inspected.

Identification of Structural Aberrations Involving BCL2 and BCL6

The presence of translocations involving MYC, BCL2 and BCL6 was determined for 49 of the DLBCL cases (FIG. 2) using commercial dual color “break-apart” probes from Abbott Molecular (Abbott Park, Ill.) on formalin fixed paraffin embedded tissue in tissue microarrays using the described method [74]. Additional fusion transcripts involving BCL2 or BCL6 were detected in these and the remaining libraries directly from the RNA-seq data using both Trans-ABySS [48] and deFuse (http://compbio.bccrc.ca/?page_id=275).

Analysis of Impact of COO and Mutation Status on Outcome in DLBCL

The analysis included only patients treated with curative intent who received at least one cycle of R-CHOP. Overall survival (OS) was calculated as the time from date of diagnosis until death from any cause. Patients were censored at the time they were last known to be alive. OS was assessed using the Kaplan-Meier method and the log rank test was used for comparison between groups. Data were analysed using SPSS software (SPSS version 14.0 for Windows; SPSS Inc, Chicago, Ill.).

TABLE 1 Overview of cSNVs and confirmed somatic mutations in most frequently mutated genes. Somatic Skew cSNVs (M, (RNA- WT, Cases Total seq NS T both) Gene NS S T NS S T cohort)* P (raw) q SP SP *** MLL2^(†) 16 8 17 17 8 18 10 6.85 × 10⁻⁸ 8.50 × 10⁻⁷ 0.834 14.4 WT TNFRSF14^(G†) 7 1 7 8 1 7 11 6.85 × 10⁻⁸ 8.50 × 10⁻⁷ 7.52 118 both SGK1^(G†) 18 6 6 37 10 6 9 6.85 × 10⁻⁸ 8.50 × 10⁻⁷ 19.5 61.7 — BCL10^(†) 2 0 4 3 0 4 4 6.85 × 10⁻⁸ 8.50 × 10⁻⁷ 3.62 112 WT GNA13^(G†) 21 1 2 33 1 2 5 6.85 × 10⁻⁸ 8.50 × 10⁻⁷ 24.1 25.7 both TP53^(G†) 20 2 1 23 3 1 22 6.85 × 10⁻⁸ 8.50 × 10⁻⁷ 15.6 14.1 both EZH2^(G†) 33 0 0 33 0 0 33 6.85 × 10⁻⁸ 8.50 × 10⁻⁷ 11.4 0.00 both BTG2^(†) 12 6 1 14 6 1 2 6.85 × 10⁻⁸ 8.50 × 10⁻⁷ 23.9 35.1 — BCL2^(G†) 42 45 0 96 105 0 43 9.35 × 10⁻⁸ 8.50 × 10⁻⁷ 3.78 0.00 M BCL6^(†)** 11 2 0 12 2 0 2 9.35 × 10⁻⁸ 8.50 × 10⁻⁷ 0.175 0.00 M CIITA^(†)** 5 3 0 6 3 0 2 9.35 × 10⁻⁸ 8.50 × 10⁻⁷ 0.086 0.00 FAS^(†) 2 0 4 3 0 4 2 1.52 × 10⁻⁷ 1.17 × 10⁻⁶ 2.54 66.5 WT BTG1^(†) 11 6 2 11 7 2 10 1.52 × 10⁻⁷ 1.17 × 10⁻⁶ 17.5 52.5 both MEF2B^(G†) 20 2 0 20 2 0 10 2.05 × 10⁻⁷ 1.47 × 10⁻⁶ 14.2 0.00 M IRF8^(†) 11 5 3 14 5 3 3 4.55 × 10⁻⁷ 3.03 × 10⁻⁶ 8.82 28.2 WT TMEM30A^(†) 1 0 4 1 0 4 4 6.06 × 10⁻⁷ 3.79 × 10⁻⁶ 0.785 65.0 WT CD58^(†) 2 0 3 2 0 3 2 2.42 × 10⁻⁶ 1.43 × 10⁻⁵ 2.29 69.2 — KLHL6^(†) 10 2 2 12 2 2 4 1.00 × 10⁻⁵ 5.26 × 10⁻⁵ 5.42 16.4 — MYD88^(A†) 13 2 0 14 2 0 9 1.00 × 10⁻⁵ 5.26 × 10⁻⁵ 12.4 0.00 WT CD70^(†) 5 0 1 5 0 2 3 1.70 × 10⁻⁵ 8.48 × 10⁻⁵ 7.08 44.0 — CD79B^(A†) 7 2 1 9 2 1 5 2.00 × 10⁻⁵ 9.52 × 10⁻⁵ 10.9 18.3 M CCND3^(†) 7 1 2 7 1 2 6 2.80 × 10⁻⁵ 1.27 × 10⁻⁴ 6.55 36.3 WT CREBBP^(†) 20 7 4 24 7 4 9 1.00 × 10⁻⁴ 4.35 × 10⁻⁴ 2.72 6.04 both HIST1H1C^(†) 9 0 0 10 0 0 6 1.80 × 10⁻⁴ 7.50 × 10⁻⁴ 11.9 0.00 both B2M^(†) 7 0 0 7 0 0 4 3.90 × 10⁻⁴ 1.56 × 10⁻³ 16.6 0.00 WT ETS1^(†) 10 1 0 10 1 0 4 4.10 × 10⁻⁴ 1.58 × 10⁻³ 5.76 0.00 WT CARD11^(†) 14 3 0 14 3 0 3 1.90 × 10⁻³ 7.04 × 10⁻³ 3.37 0.00 both FAT2^(†)** 2 1 0 2 1 0 2 6.30 × 10⁻³ 2.25 × 10⁻² 0.128 0.00 — IRF4^(†)** 9 4 0 26 5 0 5 7.00 × 10⁻³ 2.41 × 10⁻² 0.569 0.00 both FOXO1^(†) 8 4 0 10 4 0 4 7.60 × 10³ 2.53 × 10⁻² 4.02 0.00 — STAT3 9 0 0 9 0 0 4 2.19 × 10⁻² 6.08 × 10⁻² — — both RAPGEF1 8 3 0 10 3 0 3 2.98 × 10⁻² 7.45 × 10⁻² — — WT ABCA7 12 3 0 15 3 0 2 7.76 × 10⁻² 1.67 × 10⁻¹ — — WT RNF213 10 8 0 10 8 0 2 7.87 × 10⁻² 1.67 × 10⁻¹ — — — MUC16 17 12 0 39 25 0 2 8.32 × 10⁻² 1.73 × 10⁻¹ — — — HDAC7 8 4 0 8 4 0 2 8.94 × 10⁻² 1.82 × 10⁻¹ — — WT PRKDC 7 3 0 7 4 0 2 1.06 × 10⁻¹ 2.05 × 10⁻¹ — — — SAMD9 9 2 0 9 2 0 2 1.79 × 10⁻¹ 3.01 × 10⁻¹ — — — TAF1 10 0 0 10 0 0 2 3.03 × 10⁻¹ 4.74 × 10⁻¹ — — — PIM1 20 19 0 33 34 0 11 3.40 × 10⁻¹ 5.23 × 10⁻¹ — — WT COL4A2 8 2 0 8 2 0 2 7.64 × 10⁻¹ 8.99 × 10⁻¹ — — — EP300 8 7 1 8 7 1 3 9.54 × 10⁻¹ 1.00 — — WT

Individual cases with nonsynonymous (NS), synonymous (S) and truncating (T) mutations and total number of mutations of each class is shown separately as some genes contained multiple mutations in the same case. The P values indicated in bold are the upper limit on the P value for that gene determined with the approach described by Greenman et al (see Methods) [19], q is the Benjamini-corrected q value, and NS, SP and T SP refer to selective pressure estimates from this model for the acquisition of nonsynonymous or truncating mutations, respectively. †genes significant at an FDR of 0.03. SNVs in BCL2 and previously confirmed hot spot mutations in EZH2 and CD79B are likely somatic in these samples based on published observations of others. *Additional somatic mutations identified in larger cohorts and insertion/deletion mutations are not included in this total. ** Selective pressure estimates are both <1 indicating purifying selection rather than positive selection acting on this gene. *** “both” indicates we observed separate cases in which skewed expression was seen but where this skew was not consistent for the mutant or wild-type allele. Genes with a superscript of either A or G were found to have mutations significantly enriched in ABC or GCB cases, respectively (P<0.05, Fisher Exact test).

TABLE 2 Summary of types of MLL2 somatic mutations. Sample Type FL DLBCL DLBCL cell-line Centroblast Truncation 18 4 7 0 Indel with 22 8 6 0 frameshift Splice site 4 2 0 0 SNV 3 2 2 0 Any mutation 31/35 12/37 10/17 0/8 (number of cases) Percentage 89% 32% 59% 0%

TABLE 3 Mutations in selected B-cell NHL biomarkers from exome and genome sequencing. Gene Detection Base Total cSNVs symbol Ensembl id method change Annotation in gene ABCA7 ENSG00000064687 genome G > A E1322K 13 ABCA7 ENSG00000064687 RNA-seq C > T S268L 13 B2M ENSG00000166710 RNA-seq T > A Y86N 12 B2M ENSG00000166710 RNA-seq T > G M1R 12 B2M ENSG00000166710 RNA-seq A > T M1L 12 B2M ENSG00000166710 genome T > A L12Q 12 BCL10 ENSG00000142867 genome A > C L225* 4 BCL10 ENSG00000142867 genome T > A T229S 4 BCL10 ENSG00000142867 genome G > A S227L 4 BCL10 ENSG00000142867 RNA-seq G > C S136* 4 BCL10 ENSG00000142867 RNA-seq T > A R135* 4 BCL10 ENSG00000142867 RNA-seq T > A K146* 4 BCL10 ENSG00000142867 RNA-seq T > A L225F 4 BCL2 ENSG00000171791 exome C > T A2T 42 BCL2 ENSG00000171791 exome G > C H3D 42 BCL2 ENSG00000171791 RNA-seq C > A R6I 42 BCL2 ENSG00000171791 RNA-seq G > A P57S 42 BCL2 ENSG00000171791 RNA-seq C > T V35M 42 BCL2 ENSG00000171791 RNA-seq A > C M16R 42 BCL2 ENSG00000171791 RNA-seq A > G F104L 42 BCL2 ENSG00000171791 RNA-seq G > A A131V 42 BCL2 ENSG00000171791 RNA-seq C > T A61T 42 BCL2 ENSG00000171791 RNA-seq C > T A2T 42 BCL2 ENSG00000171791 RNA-seq T > A Y28F 42 BCL2 ENSG00000171791 RNA-seq G > A A60V 42 BCL2 ENSG00000171791 RNA-seq G > A L86F 42 BCL2 ENSG00000171791 RNA-seq A > G F49S 42 BCL2 ENSG00000171791 RNA-seq A > C H20Q 42 BCL2 ENSG00000171791 RNA-seq C > T R146K 42 BCL2 ENSG00000171791 RNA-seq C > G E135D 42 BCL2 ENSG00000171791 RNA-seq C > T G47D 42 BCL2 ENSG00000171791 RNA-seq T > A N11Y 42 BCL2 ENSG00000171791 RNA-seq C > T D31N 42 BCL2 ENSG00000171791 RNA-seq G > A A37V 42 BCL2 ENSG00000171791 RNA-seq C > T R129H 42 BCL2 ENSG00000171791 RNA-seq T > C M16V 42 BCL2 ENSG00000171791 RNA-seq G > A P59L 42 BCL2 ENSG00000171791 RNA-seq G > C L119V 42 BCL2 ENSG00000171791 RNA-seq A > T M16K 42 BCL2 ENSG00000171791 RNA-seq T > A T125S 42 BCL2 ENSG00000171791 RNA-seq G > A T74I 42 BCL2 ENSG00000171791 RNA-seq A > G S51P 42 BCL2 ENSG00000171791 RNA-seq C > A K17N 42 BCL2 ENSG00000171791 RNA-seq C > A G5V 42 BCL2 ENSG00000171791 RNA-seq G > A P59S 42 BCL2 ENSG00000171791 RNA-seq G > C P57A 42 BCL2 ENSG00000171791 RNA-seq T > C D34G 42 BCL2 ENSG00000171791 RNA-seq T > C I48V 42 BCL2 ENSG00000171791 RNA-seq G > C A60G 42 BCL2 ENSG00000171791 RNA-seq G > C N11K 42 BCL2 ENSG00000171791 RNA-seq T > C T69A 42 BCL2 ENSG00000171791 RNA-seq C > T A76T 42 BCL2 ENSG00000171791 RNA-seq G > A A60V 42 BCL2 ENSG00000171791 RNA-seq A > C H20Q 42 BCL2 ENSG00000171791 RNA-seq A > C S167A 42 BCL2 ENSG00000171791 RNA-seq G > A T187I 42 BCL2 ENSG00000171791 RNA-seq C > T S87N 42 BCL2 ENSG00000171791 RNA-seq A > T H20Q 42 BCL2 ENSG00000171791 RNA-seq C > G E13D 42 BCL2 ENSG00000171791 RNA-seq A > G V156A 42 BCL2 ENSG00000171791 RNA-seq G > C F104L 42 BCL2 ENSG00000171791 RNA-seq T > C N172S 42 BCL2 ENSG00000171791 RNA-seq A > G S50P 42 BCL2 ENSG00000171791 RNA-seq G > A P59L 42 BCL2 ENSG00000171791 RNA-seq G > A P59S 42 BCL2 ENSG00000171791 RNA-seq C > A R107L 42 BCL2 ENSG00000171791 RNA-seq A > G Y21H 42 BCL2 ENSG00000171791 RNA-seq T > C Q52R 42 BCL2 ENSG00000171791 RNA-seq G > C T7R 42 BCL2 ENSG00000171791 RNA-seq C > T E165K 42 BCL2 ENSG00000171791 RNA-seq G > A A80V 42 BCL2 ENSG00000171791 RNA-seq C > T R146K 42 BCL2 ENSG00000171791 RNA-seq A > G F49L 42 BCL2 ENSG00000171791 RNA-seq A > C F49C 42 BCL2 ENSG00000171791 RNA-seq C > G K17N 42 BCL2 ENSG00000171791 RNA-seq G > A P65S 42 BCL2 ENSG00000171791 RNA-seq G > T A60D 42 BCL2 ENSG00000171791 RNA-seq G > T S51Y 42 BCL2 ENSG00000171791 RNA-seq G > A P71S 42 BCL2 ENSG00000171791 RNA-seq G > A A43V 42 BCL2 ENSG00000171791 RNA-seq G > A P59S 42 BCL2 ENSG00000171791 RNA-seq C > T G27D 42 BCL2 ENSG00000171791 RNA-seq G > C A131G 42 BCL2 ENSG00000171791 RNA-seq C > T S87N 42 BCL2 ENSG00000171791 RNA-seq A > T L169Q 42 BCL2 ENSG00000171791 RNA-seq G > A A131V 42 BCL2 ENSG00000171791 RNA-seq C > A A45S 42 BCL2 ENSG00000171791 RNA-seq C > T A60T 42 BCL2 ENSG00000171791 RNA-seq T > G T69P 42 BCL2 ENSG00000171791 RNA-seq G > C S117R 42 BCL2 ENSG00000171791 RNA-seq A > G F49L 42 BCL2 ENSG00000171791 RNA-seq C > T G47D 42 BCL2 ENSG00000171791 RNA-seq C > T V66I 42 BCL2 ENSG00000171791 RNA-seq G > C P46A 42 BCL2 ENSG00000171791 RNA-seq G > A P59S 42 BCL2 ENSG00000171791 RNA-seq G > C P59A 42 BCL2 ENSG00000171791 RNA-seq G > C P46A 42 BCL2 ENSG00000171791 RNA-seq G > A A131V 42 BCL2 ENSG00000171791 RNA-seq T > A Y9F 42 BCL2 ENSG00000171791 RNA-seq A > G V159A 42 BCL2 ENSG00000171791 RNA-seq G > A T7I 42 BCL2 ENSG00000171791 RNA-seq G > A P53S 42 BCL2 ENSG00000171791 RNA-seq G > C S87R 42 BCL2 ENSG00000171791 RNA-seq G > T T7K 42 BCL2 ENSG00000171791 RNA-seq C > T R164Q 42 BCL2 ENSG00000171791 RNA-seq G > A T7I 42 BCL2 ENSG00000171791 RNA-seq T > A I48F 42 BCL2 ENSG00000171791 RNA-seq T > C Y21C 42 BCL2 ENSG00000171791 RNA-seq T > A T132S 42 BCL2 ENSG00000171791 RNA-seq T > C N143S 42 BCL2 ENSG00000171791 RNA-seq G > A A60V 42 BCL2 ENSG00000171791 RNA-seq G > A A60V 42 BCL2 ENSG00000171791 RNA-seq T > G Y108S 42 BCL6 ENSG00000113916 genome C > T A587T 11 BCL6 ENSG00000113916 RNA-seq C > T A587T 11 BTG1 ENSG00000133639 genome G > C L94V 13 BTG1 ENSG00000133639 RNA-seq G > A P58L 13 BTG1 ENSG00000133639 RNA-seq C > G Q36H 13 BTG1 ENSG00000133639 RNA-seq G > A H2Y 13 BTG1 ENSG00000133639 RNA-seq C > G Q36H 13 BTG1 ENSG00000133639 RNA-seq A > T C149* 13 BTG1 ENSG00000133639 RNA-seq C > T R27H 13 BTG1 ENSG00000133639 RNA-seq C > G A49P 13 BTG1 ENSG00000133639 RNA-seq G > C Q38E 13 BTG1 ENSG00000133639 RNA-seq C > G E46D 13 BTG2 ENSG00000159388 RNA-seq C > A A45E 13 BTG2 ENSG00000159388 RNA-seq G > A A45T 13 CARD11 ENSG00000198286 exome C > G E86Q; E93Q; E110Q 14 CARD11 ENSG00000198286 exome A > G L244P; L251P; L268P 14 CARD11 ENSG00000198286 RNA-seq T > C Q364R; Q371R; Q388R 14 CARD11 ENSG00000198286 RNA-seq A > T M353K; M360K; M377K 14 CARD11 ENSG00000198286 RNA-seq A > T F123I; F130I; F147I 14 CARD11 ENSG00000198286 RNA-seq A > T F108I; F115I; F132I 14 CARD11 ENSG00000198286 RNA-seq C > T D394N; D401N; D418N 14 CARD11 ENSG00000198286 RNA-seq A > C Y333D; Y340D; Y357D 14 CARD11 ENSG00000198286 RNA-seq A > C N230K; N237K; N254K 14 CARD11 ENSG00000198286 RNA-seq C > T D223N; D230N; D247N 14 CARD11 ENSG00000198286 RNA-seq T > G Q242P; Q249P; Q266P 14 CARD11 ENSG00000198286 RNA-seq A > C F123C; F130C; F147C 14 CARD11 ENSG00000198286 RNA-seq T > G Q242P; Q249P; Q266P 14 CARD11 ENSG00000198286 RNA-seq C > T G116D; G123D; G140D 14 CCND3 ENSG00000112576 RNA-seq G > A P234L; P280L; P284L 10 CCND3 ENSG00000112576 RNA-seq G > A Q226*; Q272*; Q276* 10 CCND3 ENSG00000112576 RNA-seq G > A Q226*; Q272*; Q276* 10 CCND3 ENSG00000112576 RNA-seq A > C I240R; I286R; I290R 10 CCND3 ENSG00000112576 RNA-seq A > T V237D; V283D; V287D 10 CCND3 ENSG00000112576 RNA-seq T > G T233P; T279P; T283P 10 CD58 ENSG00000116815 genome G > A Q141* 6 CD58 ENSG00000116815 RNA-seq C > A C131F 6 CD70 ENSG00000125726 exome A > C L60R 9 CD70 ENSG00000125726 RNA-seq A > G F186S 9 CD70 ENSG00000125726 RNA-seq C > G G66R 9 CD79B ENSG00000007312 RNA-seq T > G Y92S; Y196S; Y197S 8 CD79B ENSG00000007312 RNA-seq A > G Y92H; Y196H; Y197H 8 CD79B ENSG00000007312 RNA-seq T > A Y92F; Y196F; Y197F 8 CD79B ENSG00000007312 RNA-seq A > G Y92H; Y196H; Y197H 8 CD79B ENSG00000007312 RNA-seq T > C Y92C; Y196C; Y197C 8 CIITA ENSG00000179583 exome A > T D748V; D777V 12 CIITA ENSG00000179583 RNA-seq T > A L810Q; L839Q 12 COL4A2 ENSG00000134871 genome G > A G441D; G447D 8 COL4A2 ENSG00000134871 RNA-seq G > A G97E 8 CREBBP ENSG00000005339 exome C > T E1012K; E1042K 23 CREBBP ENSG00000005339 exome A > G Y71H; Y1482H; Y1512H 23 CREBBP ENSG00000005339 RNA-seq C > T S25N; S1436N; S1466N 23 CREBBP ENSG00000005339 RNA-seq A > T L88Q; L1499Q; L1529Q 23 CREBBP ENSG00000005339 RNA-seq A > G Y92H; Y1503H; Y1533H 23 CREBBP ENSG00000005339 RNA-seq G > C P77R; P1488R; P1518R 23 CREBBP ENSG00000005339 RNA-seq A > G L88P; L1499P; L1529P 23 CREBBP ENSG00000005339 RNA-seq G > A R35C; R1446C; R1476C 23 CREBBP ENSG00000005339 RNA-seq A > T Y71N; Y1482N; Y1512N 23 CREBBP ENSG00000005339 RNA-seq T > C M1625V; M1655V 23 CREBBP ENSG00000005339 genome G > A Q1104*; Q1134* 23 EP300 ENSG00000100393 RNA-seq T > A Y1467N 10 EP300 ENSG00000100393 RNA-seq T > C Y1467H 10 EP300 ENSG00000100393 RNA-seq G > A A1498T 10 EP300 ENSG00000100393 genome T > C L415P 10 ETS1 ENSG00000134954 RNA-seq G > A L23F 12 ETS1 ENSG00000134954 RNA-seq G > A L23F 12 ETS1 ENSG00000134954 RNA-seq C > G E22D 12 ETS1 ENSG00000134954 RNA-seq T > C M1V 12 ETS1 ENSG00000134954 genome G > C T12S 12 EZH2 ENSG00000106462 genome G > C A638G; A682G 33 EZH2 ENSG00000106462 RNA-seq G > A A648V; A692V 33 EZH2 ENSG00000106462 exome T > G Y602S; Y646S 33 EZH2 ENSG00000106462 genome T > A Y602F; Y646F 33 EZH2 ENSG00000106462 exome A > G Y602H; Y646H 33 EZH2 ENSG00000106462 RNA-seq T > A Y602F; Y646F 33 EZH2 ENSG00000106462 RNA-seq T > G Y602S; Y646S 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq A > G Y602H; Y646H 33 EZH2 ENSG00000106462 RNA-seq A > G Y602H; Y646H 33 EZH2 ENSG00000106462 RNA-seq T > A Y602F; Y646F 33 EZH2 ENSG00000106462 RNA-seq A > G Y602H; Y646H 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq T > A Y602F; Y646F 33 EZH2 ENSG00000106462 RNA-seq T > G Y602S; Y646S 33 EZH2 ENSG00000106462 RNA-seq A > G Y602H; Y646H 33 EZH2 ENSG00000106462 RNA-seq T > A Y602F; Y646F 33 EZH2 ENSG00000106462 RNA-seq A > G Y602H; Y646H 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq T > A Y602F; Y646F 33 EZH2 ENSG00000106462 RNA-seq T > G Y602S; Y646S 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq T > A Y602F; Y646F 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 EZH2 ENSG00000106462 RNA-seq T > A Y602F; Y646F 33 EZH2 ENSG00000106462 RNA-seq A > T Y602N; Y646N 33 FAS ENSG00000026103 exome C > T Q255*; Q276*; Q303* 6 FAS ENSG00000026103 RNA-seq T > G Y211*; Y232*; Y259* 6 FAS ENSG00000026103 genome G > C V224L; V245L; V272L 6 FAS ENSG00000026103 genome A > G D244G; D265G; D292G 6 FAT2 ENSG00000086570 exome C > T D1287N 2 FAT2 ENSG00000086570 exome C > T G994R 2 FOXO1 ENSG00000150907 RNA-seq C > T S203N 10 FOXO1 ENSG00000150907 RNA-seq T > C M1V 10 FOXO1 ENSG00000150907 RNA-seq G > A T24I 10 FOXO1 ENSG00000150907 RNA-seq G > T S193R 10 FOXO1 ENSG00000150907 RNA-seq T > C T24A 10 GNA13 ENSG00000120063 RNA-seq G > A L296F 22 GNA13 ENSG00000120063 RNA-seq T > C K292R 22 GNA13 ENSG00000120063 RNA-seq T > C T262A 22 GNA13 ENSG00000120063 RNA-seq A > G *378R 22 GNA13 ENSG00000120063 RNA-seq T > A K42* 22 GNA13 ENSG00000120063 RNA-seq T > G H345P 22 GNA13 ENSG00000120063 RNA-seq T > C T203A 22 GNA13 ENSG00000120063 RNA-seq G > A S31F 22 GNA13 ENSG00000120063 genome A > T I158K 22 HDAC7 ENSG00000061273 genome G > A S155F; S194F 9 HDAC7 ENSG00000061273 RNA-seq C > T A786T; A788T; A825T 9 HIST1H1C ENSG00000187837 genome G > C A185G 10 HIST1H1C ENSG00000187837 genome C > G A180P 10 HIST1H1C ENSG00000187837 RNA-seq G > A P118S 10 HIST1H1C ENSG00000187837 RNA-seq C > G V132L 10 HIST1H1C ENSG00000187837 RNA-seq G > C L107V 10 HIST1H1C ENSG00000187837 RNA-seq C > T E74K 10 HIST1H1C ENSG00000187837 genome C > G G103A 10 IKZF3 ENSG00000161405 RNA-seq T > G N73T; N160T 7 IRF4 ENSG00000137265 RNA-seq G > C S18T 9 IRF4 ENSG00000137265 RNA-seq C > G L40V 9 IRF4 ENSG00000137265 RNA-seq A > G I32V 9 IRF4 ENSG00000137265 RNA-seq A > G N2S 9 IRF4 ENSG00000137265 RNA-seq C > A Q60K 9 IRF4 ENSG00000137265 RNA-seq C > G S18R 9 IRF4 ENSG00000137265 RNA-seq G > C Q60H 9 IRF4 ENSG00000137265 RNA-seq A > C S48R 9 IRF4 ENSG00000137265 RNA-seq C > A S48R 9 IRF8 ENSG00000140968 genome T > G S55A 14 IRF8 ENSG00000140968 genome G > C S34T 14 IRF8 ENSG00000140968 RNA-seq A > T *427L 14 KLHL6 ENSG00000172578 genome C > G S83T; S94T 13 KLHL6 ENSG00000172578 RNA-seq G > C T53S; T64S 13 KLHL6 ENSG00000172578 RNA-seq A > T L45*; L56* 13 KLHL6 ENSG00000172578 RNA-seq G > A T53I; T64I 13 KLHL6 ENSG00000172578 RNA-seq G > C L54V; L65V 13 MEF2B ENSG00000064489 exome T > C Y69C 20 MEF2B ENSG00000064489 RNA-seq T > A D83V 20 MEF2B ENSG00000064489 RNA-seq T > A D83V 20 MEF2B ENSG00000064489 RNA-seq T > A D83V 20 MEF2B ENSG00000064489 RNA-seq A > C L67R 20 MEF2B ENSG00000064489 RNA-seq A > G Y69H 20 MEF2B ENSG00000064489 RNA-seq T > A D83V 20 MEF2B ENSG00000064489 RNA-seq T > G D83A 20 MEF2B ENSG00000064489 RNA-seq T > A N81Y 20 MEF2B ENSG00000064489 genome G > T N81K 20 MLL2 ENSG00000167548 genome G > A Q3391* 29 MLL2 ENSG00000167548 RNA-seq C > G A4607P 29 MLL2 ENSG00000167548 RNA-seq C > T R2547H 29 MLL2 ENSG00000167548 RNA-seq G > A R2250* 29 MLL2 ENSG00000167548 RNA-seq G > A P3583S 29 MLL2 ENSG00000167548 RNA-seq G > A R4634C 29 MLL2 ENSG00000167548 RNA-seq G > A R3956* 29 MLL2 ENSG00000167548 RNA-seq G > A Q3333* 29 MLL2 ENSG00000167548 RNA-seq G > A R4921* 29 MLL2 ENSG00000167548 RNA-seq G > A R2107* 29 MLL2 ENSG00000167548 genome G > A Q3394* 29 MUC16 ENSG00000181143 genome A > G S2928P 17 MUC16 ENSG00000181143 genome T > G S1055R 17 MUC16 ENSG00000181143 genome G > T S464Y; S2725Y; S4093Y; S8460Y 17 MYD88 ENSG00000172936 RNA-seq C > G S206C 14 MYD88 ENSG00000172936 RNA-seq T > C L252P 14 MYD88 ENSG00000172936 RNA-seq T > C L252P 14 MYD88 ENSG00000172936 RNA-seq T > C L252P 14 MYD88 ENSG00000172936 RNA-seq T > C L252P 14 MYD88 ENSG00000172936 RNA-seq T > C L252P 14 MYD88 ENSG00000172936 RNA-seq C > G S206C 14 MYD88 ENSG00000172936 RNA-seq G > A S230N 14 MYD88 ENSG00000172936 genome G > A S230N 14 PIM1 ENSG00000137193 RNA-seq C > G L164V; L255V 21 PIM1 ENSG00000137193 RNA-seq C > G L164V; L255V 21 PIM1 ENSG00000137193 RNA-seq C > G L25V; L116V 21 PIM1 ENSG00000137193 RNA-seq C > T L164F; L255F 21 PIM1 ENSG00000137193 RNA-seq G > C E181D; E272D 21 PIM1 ENSG00000137193 RNA-seq G > A S97N; S188N 21 PIM1 ENSG00000137193 RNA-seq G > A S97N; S188N 21 PIM1 ENSG00000137193 RNA-seq G > C E79D; E170D 21 PIM1 ENSG00000137193 RNA-seq G > C K24N; K115N 21 PIM1 ENSG00000137193 RNA-seq C > G S146R; S237R 21 PIM1 ENSG00000137193 RNA-seq G > C Q37H; Q128H 21 PIM1 ENSG00000137193 RNA-seq C > G S146R; S237R 21 PIM1 ENSG00000137193 RNA-seq C > T L2F; L93F 21 PIM1 ENSG00000137193 RNA-seq C > G L2V; L93V 21 PIM1 ENSG00000137193 RNA-seq G > C Q37H; Q128H 21 PLCG2 ENSG00000197943 exome C > A S16R 7 PRKDC ENSG00000121031 genome A > C F1854V 7 PRKDC ENSG00000121031 RNA-seq A > C F3973V; F4004V 7 RAPGEF1 ENSG00000107263 RNA-seq C > T S53N; S284N; S358N; S375N; S376N 8 RAPGEF1 ENSG00000107263 RNA-seq A > T Y265N; Y496N; Y570N; Y587N; Y588N 8 RAPGEF1 ENSG00000107263 RNA-seq C > G V16L; V297L; V528L; V602L; V619L; V620L 8 RAPGEF1 ENSG00000107263 genome A > T M250K; M481K; M555K; M572K; M573K 8 RFTN1 ENSG00000131378 exome C > A S224I 6 RFTN1 ENSG00000131378 RNA-seq G > A P205S 6 RNF213 ENSG00000173821 genome T > A N2194K 11 RNF213 ENSG00000173821 RNA-seq G > A R2286Q 11 SAMD9 ENSG00000205413 genome T > A N615Y 11 SAMD9 ENSG00000205413 RNA-seq A > G I1578T 11 SGK1 ENSG00000118515 exome C > G A105P; A115P; A129P; A210P 20 SGK1 ENSG00000118515 RNA-seq T > C R21G; R31G; R45G; R126G 20 SGK1 ENSG00000118515 RNA-seq G > T A115E; A125E; A139E; A220E 20 SGK1 ENSG00000118515 RNA-seq G > T H153Q; H163Q; H177Q; H258Q 20 SGK1 ENSG00000118515 RNA-seq G > C A193G; A203G; A217G; A298G 20 SGK1 ENSG00000118515 RNA-seq A > T N34K; N44K; N58K; N139K 20 SGK1 ENSG00000118515 RNA-seq G > C F113L; F123L; F137L; F218L 20 SGK1 ENSG00000118515 RNA-seq C > G S242T; S252T; S266T; S347T 20 SGK1 ENSG00000118515 RNA-seq G > A P67S; P77S; P91S; P172S 20 SGK1 ENSG00000118515 RNA-seq T > A K19M; K29M; K43M; K124M 20 SGK1 ENSG00000118515 RNA-seq G > A Q30*; Q40*; Q54*; Q135* 20 SGK1 ENSG00000118515 RNA-seq G > A T5I 20 SGK1 ENSG00000118515 RNA-seq C > A E136*; E146*; E160*; E241* 20 SGK1 ENSG00000118515 RNA-seq G > A P65S; P75S; P89S; P170S 20 SGK1 ENSG00000118515 RNA-seq G > A P63S; P73S; P87S; P168S 20 SGK1 ENSG00000118515 RNA-seq C > A R22M; R32M; R46M; R127M 20 SGK1 ENSG00000118515 RNA-seq G > T T229N; T239N; T253N; T334N 20 SGK1 ENSG00000118515 RNA-seq C > G R211T; R221T; R235T; R316T 20 SGK1 ENSG00000118515 genome C > T C183Y; C193Y; C207Y; C288Y 20 SGK1 ENSG00000118515 genome G > T R6S 20 SGK1 ENSG00000118515 genome C > A E338*; E348*; E362*; E443* 20 SGK1 ENSG00000118515 genome G > A P81L; P91L; P105L; P186L 20 SGK1 ENSG00000118515 genome G > A P11L 20 STAT3 ENSG00000168610 exome G > C S614R 9 STAT3 ENSG00000168610 RNA-seq A > T N567K 9 STAT3 ENSG00000168610 RNA-seq C > T E616K 9 STAT3 ENSG00000168610 RNA-seq C > T D566N 9 STAT6 ENSG00000166888 exome G > T Q286K 6 STAT6 ENSG00000166888 RNA-seq T > C D419G 6 TAF1 ENSG00000147133 genome T > C L1000P; L1021P 10 TAF1 ENSG00000147133 RNA-seq T > C F1047S; F1068S 10 TMEM30A ENSG00000112697 genome A > T D155E; D191E 4 TMEM30A ENSG00000112697 genome A > C Y157*; Y193* 4 TMEM30A ENSG00000112697 RNA-seq G > T S280*; S316* 4 TMEM30A ENSG00000112697 RNA-seq G > A R254*; R290* 4 TMEM30A ENSG00000112697 RNA-seq C > T W281*; W317* 4 TNFRSF14 ENSG00000157873 RNA-seq C > T W12* 14 TNFRSF14 ENSG00000157873 RNA-seq G > T C57* 14 TNFRSF14 ENSG00000157873 RNA-seq G > C S112C 14 TNFRSF14 ENSG00000157873 RNA-seq C > T W201* 14 TNFRSF14 ENSG00000157873 RNA-seq T > A N110Y 14 TNFRSF14 ENSG00000157873 RNA-seq C > T W12* 14 TNFRSF14 ENSG00000157873 RNA-seq G > A Q95* 14 TNFRSF14 ENSG00000157873 RNA-seq A > G C53R 14 TNFRSF14 ENSG00000157873 RNA-seq G > T Y47* 14 TNFRSF14 ENSG00000157873 genome C > T W7* 14 TNFRSF14 ENSG00000157873 genome C > T G60D 14 TP53 ENSG00000141510 RNA-seq C > T V50M; V143M 21 TP53 ENSG00000141510 RNA-seq A > C C83G; C176G 21 TP53 ENSG00000141510 RNA-seq T > C Y127C; Y220C 21 TP53 ENSG00000141510 RNA-seq A > T Y112N; Y205N 21 TP53 ENSG00000141510 RNA-seq A > C Y107D 21 TP53 ENSG00000141510 RNA-seq T > C Y141C; Y234C 21 TP53 ENSG00000141510 RNA-seq A > T Y141N; Y234N 21 TP53 ENSG00000141510 RNA-seq G > A R155W; R248W 21 TP53 ENSG00000141510 RNA-seq A > C Y107D 21 TP53 ENSG00000141510 RNA-seq A > C S122R; S215R 21 TP53 ENSG00000141510 RNA-seq A > C Y107D 21 TP53 ENSG00000141510 RNA-seq G > A R155W; R248W 21 TP53 ENSG00000141510 RNA-seq C > A G262V 21 TP53 ENSG00000141510 RNA-seq A > G F41L; F134L 21 TP53 ENSG00000141510 RNA-seq C > T R65H; R158H 21 TP53 ENSG00000141510 RNA-seq A > C Y33D; Y126D 21 TP53 ENSG00000141510 RNA-seq C > T G152D; G245D 21 TP53 ENSG00000141510 RNA-seq T > C T18A 21 TP53 ENSG00000141510 RNA-seq C > A C83F; C176F 21 TP53 ENSG00000141510 RNA-seq T > A K319* 21 TP53 ENSG00000141510 RNA-seq G > A R155W; R248W 21 TP53 ENSG00000141510 RNA-seq T > C Y141C; Y234C 21 TP53 ENSG00000141510 RNA-seq T > A I255F 21 TP53 ENSG00000141510 RNA-seq G > A P278L 21 TP53 ENSG00000141510 RNA-seq T > A M144L; M237L 21

TABLE 4 Mutation hotspots in genes identified using RNA-seq. Number of Distinct Codon Samples mutations Gene Name 602; 646 30 4 EZH2 83^(§) 9 2 MEF2B 69^(§) 4 2 MEF2B 81^(§) 2 2 MEF2B 1482^(§)  3 2 CREBBP 1499^(§)  2 2 CREBBP 1467^(§)  2 2 EP300 287^(§)  2 1 HLA-C 1 8 5 BCL7A ^(‡) 206^(§)  4 1 MYD88 ^(‡) 230^(§)  2 1 MYD88 ^(‡) 252^(§)  6 1 MYD88 ^(‡) 59  7 3 BCL2* 92; 196; 197 5 4 CD79B ^(‡)   73; 160^(§) 4 2 IKZF3^(‡)  164; 255^(§) 3 2 PIM1 ^(‡)  97; 188 3 2 PIM1 ^(‡) 18^(§) 3 2 IRF4 ^(‡) 587^(§)  3 2 BCL6 45^(§) 3 2 BTG2^(‡) 141; 234 3 2 TP53 ^(‡) 24^(§) 2 2 FOXO1^(‡)  1^(§) 3 3 FOXO1^(‡) 12^(§) 2 1 TNFRSF14 226^(§)  2 2 CCND3^(‡) 233^(§)  2 2 CCND3^(‡)  1^(§) 3 3 B2M^(‡) ^(§)This mutation was proven to be somatic in at least one case; that is, present in tumour DNA but absent in matched constitutional DNA. ^(‡)Not mutated in any of the fourteen genomes or exomes sequenced. *Additional hot spots in BCL2 were excluded to simplify the table. Genes indicated in bold are previously described targets of somatic mutation in lymphoma. Although known to be mutated, hot spots have not, to our knowledge, been described in BCL7A. Note that Tyr641 as previously described [13] is based on the Uniprot sequence Q15910, whereas this site corresponds to residue 602 and 646 in the Refseq annotations.

TABLE 5 Mutations affecting CREBBP or EP300 detected using RNA-seq data. EP300 Library Disease Gene Annotation position HS0841 DLBCL CREBBP E1238*; E1268* E1202 line HS0842 DLBCL CREBBP A436V A420 line HS0842 DLBCL CREBBP Q170*; Q238* not line conserved HS0806 FL CREBBP Y71H; Y1482H; Y1512H^(§) Y1446 HS1185 FL CREBBP G1411E; G1441E G1375 HS1200 FL CREBBP Y92F; Y1503F; Y1533F Y1467 HS1360 FL CREBBP R35C; R1446C; R1476C R1410 HS1361 FL CREBBP S25N; S1436N; S1466N^(§) S1400 HS0637 DLBCL CREBBP Q1104*; Q1134* Q1068 HS0641 DLBCL CREBBP L88Q; L1499Q; L1529Q^(§) L1463 HS0649 DLBCL CREBBP P77R; P1488R; P1518R^(§) P1452 HS0649 DLBCL CREBBP A687V; A717V not conserved HS0749 DLBCL CREBBP N1589K; N1619K N1552 HS0933 DLBCL CREBBP R370*; R438* R354 HS0939 DLBCL CREBBP M1625V; M1655V^(§) M1588 HS1135 DLBCL CREBBP V1342E; V1372E V1306 HS1460 DLBCL CREBBP L88P; L1499P; L1529P^(§) L1463 HS1977 DLBCL CREBBP C1283R; C1313R C1247 HS1979 DLBCL CREBBP N513S; N1978S; N2008S not conserved HS2059 DLBCL CREBBP Y71N; Y1482N; Y1512N^(§) Y1446 HS2249 DLBCL CREBBP A442T; A1907T; A1937T not conserved HS2249 DLBCL CREBBP Y92H; Y1503H; Y1533H^(§) Y1467 HS2606 DLBCL CREBBP R35C; R1446C; R1476C^(§) R1410 HS0653 DLBCL EP300 Q1904* — HS0939 DLBCL EP300 A1498T^(§) — HS1133 DLBCL EP300 L415P — HS1462 DLBCL EP300 Y1467H ^(§) — HS2049 DLBCL EP300 P925T^(‡) — HS2607 DLBCL EP300 P925T^(‡) — HS1199 FL EP300 D1485V — HS1201 FL EP300 Q1455L — HS1202 FL EP300 Y1467N ^(§) — HS0841 DLBCL EP300 Q160* — line HS0900 DLBCL EP300 R1627W — line ^(§)mutation was proven to be somatic (absent in matched constitutional DNA); ^(‡)was also found in the matched constitutional DNA (inherited variant); bold indicates mutation hot spots.

TABLE 6 Mutations in MLL2 found by targeted MLL2 resequencing. Chromosome locus Mutation Event Lymphoma Somatic status chr12: 47731299 GAG > TAG E812* FL somatic chr12: 47720827 −A Frameshift deletion FL somatic chr12: 47731577 −GCTGGAGGAGTCACCC Frameshift deletion FL somatic chr12: 47719922 TCA > TAA S2633* FL somatic chr12: 47728117 −AT Frameshift deletion FL somatic chr12: 47718602  TCA > TGA S2935* FL somatic chr12: 47706246 GAC > GTC D5257V_FYRC domain FL somatic chr12: 47706727 CGA > TGA R5097* FL somatic chr12: 47719661 CGA > TGA R2685* FL somatic chr12: 47731461  GAG > TAG E758* FL somatic chr12: 47733524 T > C SS end6 FL somatic chr12: 47729734 CAG > TAG Q1302* FL somatic chr12: 47719040  G > A SS beg34 FL somatic chr12: 47721300  CAG > TAG Q2174* FL somatic chr12: 47728117 −AT Frameshift deletion FL somatic chr12: 47707855 CAG > TAG Q4881* FL somatic chr12: 47718680  −AAGT Frameshift deletion FL somatic chr12: 47717409 CAG > TAG Q3333* FL somatic chr12: 47724315 −CA Frameshift deletion ++ FL somatic chr12: 47711008 CGA > TGA R4536* FL somatic chr12: 47734195 −GCAGCGCTG Frameshift deletion FL somatic (SSbeg5) chr12: 47711624 TGG > TGA W4377* FL somatic chr12: 47719271  G > A SS end33 FL somatic chr12: 47718918  CGA > TGA R2830* FL somatic chr12: 47713018  CAG > TAG Q3913* FL somatic chr12: 47720103  −G Frameshift deletion ++ FL somatic chr12: 47702684  CGG > TGG R5432W_SET domain FL somatic chr12: 47713509  −ACAG Frameshift deletion FL somatic chr12: 47731159  +T Frameshift insertion FL somatic chr12: 47717445  CGA > TGA R3321* FL somatic chr12: 47709482  +AT Frameshift insertion FL somatic chr12: 47714889  −G +TA Frameshift in-del FL somatic chr12: 47717767  +T Frameshift deletion FL somatic chr12: 47722866  CGA > TGA R1903* FL somatic chr12: 47720228  −C Frameshift deletion FL somatic chr12: 47704937  CGA > TGA R5282* FL undetermined chr12: 47726475  G > A SS beg16 FL undetermined chr12: 47702165  −CG +T Frameshift deletion in-del FL undetermined chr12: 47713960  CAG > TAG Q3599* FL undetermined chr12: 47713064  +T Frameshift insertion FL undetermined chr12: 47723788 −C Frameshift deletion FL undetermined chr12: 47704873 CGC > CAC R5303H_FYRC domain FL undetermined chr12: 47719320  +CGACTCT Frameshift insertion FL undetermined chr12: 47702170 −TG Frameshift deletion FL undetermined chr12: 47718081  +G Frameshift insertion FL undetermined chr12: 47704646 +G Frameshift insertion FL undetermined chr12: 47714203  +A Frameshift insertion FL undetermined chr12: 47718680  −AAGT Frameshift deletion GCB-DLBCL somatic chr12: 47726113  T > G SS end17 GCB-DLBCL somatic chr12: 47730448 +G Frameshift insertion GCB-DLBCL somatic chr12: 47724460  TAT > TAA Y1692* GCB-DLBCL somatic chr12: 47712844  CAA > TAA Q3971* GCB-DLBCL somatic chr12: 47724319  −A Frameshift deletion ABC-DLBCL somatic chr12: 47706936  CGA > CAA R5027L_FYRC domain GCB-DLBCL somatic chr12: 47723144  −ACAG Frameshift deletion GCB-DLBCL undetermined chr12: 47710329  G > A SS end42 GCB-DLBCL undetermined chr12: 47719628  CAG > TAG Q2696* GCB-DLBCL somatic chr12: 47732160  −AG Frameshift deletion GCB-DLBCL undetermined chr12: 47718251  −TA Frameshift deletion GCB-DLBCL somatic chr12: 47719327 CGA > TGA R2771* ABC-DLBCL somatic chr12: 47710444  +C Frameshift insertion ABC-DLBCL undetermined chr12: 47709214  −G Frameshift deletion GCB-DLBCL somatic chr12: 47733683  CGC > GGC R228G_PHD domain GCB-DLBCL undetermined chr12: 47719508  CAG > TAG Q2736* GCB-DLBCL^(cl) cell line chr12: 47732295 −C Frameshift deletion GCB-DLBCL^(cl) cell line chr12: 47717574  CAA > TAA Q3278* GCB-DLBCL^(cl) cell line chr12: 47717760  GAG > TAG E3216* ABC-DLBCL^(cl) cell line chr12: 47720598  +A Frameshift insertion ABC-DLBCL^(cl) cell line chr12: 47702767  TCC > TTC S5404F_SET domain GCB-DLBCL^(cl) cell line chr12: 47712865  CAG > TAG Q3964* ABC-DLBCL^(cl) cell line chr12: 47729996  −G Frameshift deletion ABC-DLBCL^(cl) cell line chr12: 47722866 CGA > TGA A1903* GCB-DLBCL^(cl) cell line chr12: 47707230  −C Frameshift deletion GCB-DLBCL^(cl) cell line chr12: 47717493 −GTTTGGCTGGGTCCCA Frameshift deletion ++ GCB-DLBCL^(cl) cell line chr12: 47734070 CAG > TAG Q211* GCB-DLBCL^(cl) cell line chr12: 47709228 GAG > TAG E4712* GCB-DLBCL^(cl) cell line chr12: 47731793 +C Frameshift insertion ++ ABC-DLBCL^(cl) cell line chr12: 47706741 TGC > TAC C5092Y_PHD domain GCB-DLBCL^(cl) cell line Additional mutations at splice sites in MLL2 detected by Trans-ABySS  chr12: 47733693 T > G SS end38 DLBCL n/a chr12: 47714115 T > G SS beg6 DLBCL n/a ++ homozygous mutations; SS Splice site mutations; *not detected by RNA-seq automated analysis; ** indels and mutations at splice sites were not part of our automated analysis of RNA-seq; n/a refers to samples for which either RNA-seq or targeted resequencing was not performed.

TABLE 7 All MEF28 mutations detected. Diagnosis and subtype Case Position Change Change (subtyping (res_id) (chromosome) (DNA) (protein) method) 03-31934 chr19: 19122543 T > A M1K FL 02-17440 chr19: 19122535 A > G K4E GCB DLBCL (GEP) 98-17403 chr19: 19122535 A > G K4E DLBCL 06-20044 chr19: 19122535^(§) A > G K4E FL 06-23741 chr19: 19122535^(§) A > G K4E FL 07-14540 chr19: 19122535 A > G K4E FL 98-14740 chr19: 19122535 A > G K4E FL 05-15463 chr19: 19122532 A > G K5E FL 03-28045 chr19: 19122523 A > G I8V DLBCL 92-59893 chr19: 19122502 A > G R15G DLBCL 02-28712 chr19: 19122492 C > T Q18* DLBCL 05-22052 chr19: 19121225 A > G K23R DLBCL 07-10201 chr19: 19121222 G > A R24Q FL SPEC1187 chr19: 19121217 T > G F26V GCB DLBCL (GEP) 06-20952 chr19: 19121195 A > C Y33S FL 03-18669 chr19: 19121153 T > C I47T DLBCL 03-33888 chr19: 19121135 G > A R53H DLBCL 01-16433 chr19: 19121093^(§) T > G L67R FL 00-15694 chr19: 19121088^(§) A > G Y69H GCB DLBCL (GEP) 05-11328 chr19: 19121088 A > G Y69H GCB DLBCL (GEP) 06-12968 chr19: 19121087^(§) T > C Y69C FL 06-18193 chr19: 19121087 T > C Y69C FL 08-10448 chr19: 19121087 T > C Y69C FL 99-30068 chr19: 19121087 T > C Y69C FL 05-11369 chr19: 19121066 −GGGGCT E74-P75- FL H76 > D 06-23851 chr19: 19121066 A > G H76R FL 07-21828 chr19: 19121064 G > A E77K DLBCL 07-30109 chr19: 19121063 A > G E77G Composite FL 06-30145 chr19: 19121052^(§) A > T N81Y GCB DLBCL (GEP) 05-23110 chr19: 19121050^(§) C > A N81K GCB DLBCL (GEP) 00-13940 chr19: 19121045 T > G D83A GCB DLBCL (IHC) 06-15922 chr19: 19121045^(§) T > G D83A GCB DLBCL (GEP) 07-23804 chr19: 19121045 T > G D83A GCB DLBCL (GEP) 00-22287 chr19: 19121045 T > A D83V GCB DLBCL (IHC) 01-18672 chr19: 19121045 T > A D83V GCB DLBCL (IHC) 02-30647 chr19: 19121045^(§) T > A D83V GCB DLBCL (GEP) 03-11110 chr19: 19121045 T > A D83V DLBCL 03-26817 chr19: 19121045 T > A D83V GCB DLBCL (GEP) 03-30438 chr19: 19121045 T > A D83V GCB DLBCL (GEP) 05-24666 chr19: 19121045 T > A D83V GCB DLBCL (GEP) 06-30025 chr19: 19121045^(§) T > A D83V GCB DLBCL (GEP) 06-33777 chr19: 19121045^(§) T > A D83V GCB DLBCL (GEP) 78-60284 chr19: 19121045 T > A D83V GCB DLBCL (IHC) 95-32814 chr19: 19121045^(§) T > A D83V GCB DLBCL (GEP) 97-10270 chr19: 19121045 T > A D83V DLBCL DB chr19: 19121045 T > A D83V GCB (cell line) DLBCL (GEP) 06-11109 chr19: 19121045 T > G D83A FL 07-20462 chr19: 19121045 T > G D83A FL 91-34915 chr19: 19121045 T > G D83A FL 03-16286 chr19: 19121045 T > C D83G FL 05-12024 chr19: 19121045 T > A D83V FL 06-22766 chr19: 19121045 T > A D83V FL 06-33903 chr19: 19121045 T > A D83V FL 89-30159 chr19: 19121045 T > A D83V FL 91-53679 chr19: 19121045 T > A D83V FL 97-23234 chr19: 19121045 T > A D83V FL 99-21548 chr19: 19121045 T > A D83V FL 01-24821 chr19: 19119600 +A L100 FL Frameshift 85-31959 chr19: 19119578 C > A E108* FL 06-16716 chr19: 19119559^(‡) C > T R114Q ABC DLBCL (GEP) 02-18484 chr19: 19119539 10bp del G121 FL Frameshift 91-53679 chr19: 19118877 −GGAA F170 FL Frameshift 08-15460 chr19: 19118875 −AAGG P169 DLBCL Frameshift 06-10398 chr19: 19118406 +GG G242 ABC Frameshift DLBCL (GEP) 06-30389 chr19: 19118365 −C P256 FL Frameshift 07-18609 chr19: 19117831 A > C S294R† FL 05-20543 chr19: 19117794 G > T R307S† ABC DLBCL (GEP) 05-14545 chr19: 19117608 A > G *369G† FL 06-23851 chr19: 19117608 A > C *369E† FL 06-12557 chr19: 19117606 C > G *369Y† FL †annotation is unique to NM_001145785, representing the longest MEF2B isoform; ^(§)was proven to be somatic (absent in matched constitutional DNA); ^(‡)was also found in the matched constitutional DNA (inherited variant).

TABLE 8 Catalogue of MEF2B cSNVs in FL and DLBCL. Amino Acid Change FL DLBCL Total % variants M1K 1 0 1 1.4 K4E^(§) 4 2 6 8.7 K5E 1 0 1 1.4 I8V 0 1 1 1.4 R15G 0 1 1 1.4 K23R 0 1 1 1.4 R24Q 1 0 1 1.4 F26V 0 1 1 1.4 Y33S 1 0 1 1.4 I47T 0 1 1 1.4 R53H 0 1 1 1.4 L67R 1 0 1 1.4 Y69C/H^(§) 4 2 6 8.7 E74-P75-H76 > D 1 0 1 1.4 H76R 1 0 1 1.4 E77K 0 1 1 1.4 N81K/Y^(§) 0 2 2 2.9 D83A/G/V^(§) 11 16 27 39.1 R114Q 0 1 1 1.4 S294Y 1 0 1 1.4 R307S 0 1 1 1.4 *369Y/E/G 3 0 3 4.3 Truncation 5 3 8 11.6 Any mutation 35 34 69 100.0 Total cases sequenced 261 292 Prevalence 13.41% 11.64% ^(§)at least one representative mutation at this position has been confirmed as a somatic mutation.

TABLE 9 All cSNVs detected in 10 DLBCL cell lines using RNA-seq data. Gene name Ensembl gene Mutation Effect (all isoforms) Cell Line HLA-C ENSG00000204525 C > G W188S; W191S OCI-Ly19 AFF1 ENSG00000172493 C > T P866P OCI-Ly7 AQR ENSG00000021776 G > C A1013G DB ASCC3L1 ENSG00000144028 T > C M387V OCI-Ly1 ASCC3L1 ENSG00000144028 T > C N313D OCI-Ly7 BCL2 ENSG00000171791 G > A N172N DB BCL2 ENSG00000171791 G > A L119L DB BCL2 ENSG00000171791 C > G R183R Karpas422 BCL2 ENSG00000171791 G > A P59L Karpas422 BCL2 ENSG00000171791 C > T G47D Karpas422 BCL2 ENSG00000171791 C > T R63R NU-DHL-1 BCL2 ENSG00000171791 C > T A2T NU-DHL-1 BCL2 ENSG00000171791 C > T L72L SU-DHL-6 BCL2 ENSG00000171791 C > T P71P SU-DHL-6 BCL2 ENSG00000171791 T > A I48F SU-DHL-6 BCL2 ENSG00000171791 T > G T69P WSU-DLCL2 BCL2 ENSG00000171791 C > G E13D WSU-DLCL2 BCL2 ENSG00000171791 G > A T187I OCI-Ly1 BCL2 ENSG00000171791 G > A S161S OCI-Ly1 BCL2 ENSG00000171791 G > A A131V OCI-Ly1 BCL2 ENSG00000171791 G > A S87S OCI-Ly1 BCL2 ENSG00000171791 C > T A85A OCI-Ly1 BCL2 ENSG00000171791 A > G F49L OCI-Ly1 BCL2 ENSG00000171791 A > G H20H OCI-Ly1 BCL2 ENSG00000171791 A > G D10D OCI-Ly1 BCL2 ENSG00000171791 C > T G5G OCI-Ly1 BCL6 ENSG00000113916 G > T A587D OCI-Ly7 BCL6 ENSG00000113916 T > G N588H OCI-Ly19 BCL7A ENSG00000110987 T > G M1R OCI-Ly1 BCL7A ENSG00000110987 C > T R29C OCI-Ly7 CARD11 ENSG00000198286 C > T D223N; D230N; D247N Karpas422 CARS ENSG00000110619 G > A H147H; H157H; H240H OCI-Ly7 CCND3 ENSG00000112576 G > A P234S; P280S; P284S NU-DHL-1 CCND3 ENSG00000112576 T > C T233A; T279A; T283A OCI-Ly7 CCND3 ENSG00000112576 C > G A239P; A285P; A289P OCI-Ly19 CENPP ENSG00000188312 G > A R141H; R182H NU-DUL-1 CREBBP ENSG00000005339 C > A E1238*; E1268* Karpas422 CREBBP ENSG00000005339 G > A A436V NU-DHL-1 CREBBP ENSG00000005339 G > A Q170*; Q238* NU-DHL-1 CSTF2T ENSG00000177613 T > A L428F DOHH-2 DBN1 ENSG00000113758 C > T R226Q; R228Q DB DDX56 ENSG00000136271 G > A L14L WSU-DLCL2 EGLN1 ENSG00000135766 T > G S166R OCI-Ly19 EZH2 ENSG00000106462 A > T Y602N; Y646N DB EZH2 ENSG00000106462 A > T Y602N; Y646N Karpas422 EZH2 ENSG00000106462 A > T Y602N; Y646N SU-DHL-6 EZH2 ENSG00000106462 T > A Y602F; Y646F WSU-DLCL2 EZH2 ENSG00000106462 A > T Y602N; Y646N OCI-Ly1 FAT4 ENSG00000196159 C > A I1760I; I3462I Karpas422 FOXO1 ENSG00000150907 T > C I10V OCI-Ly1 FOXO1 ENSG00000150907 T > A M1L OCI-Ly1 GCN1L1 ENSG00000089154 A > G L2229L OCI-Ly1 GNA13 ENSG00000120063 G > C Y89* DOHH-2 GNA13 ENSG00000120063 T > G Y308S SU-DHL-6 GNA13 ENSG00000120063 A > G F245S WSU-DLCL2 GNA13 ENSG00000120063 A > T L197Q OCI-Ly1 GNA13 ENSG00000120063 A > G I34T OCI-Ly1 GTF3C1 ENSG00000077235 C > T R403Q; R405Q OCI-Ly7 HNRNPA1 ENSG00000135486 T > G G234G OCI-Ly19 IFNGR2 ENSG00000159128 A > C I77L; I156L; I175L OCI-Ly1 IKZF3 ENSG00000161405 T > C N73S; N160S DOHH-2 IKZF3 ENSG00000161405 A > C L75R; L162R NU-DUL-1 LSP1 ENSG00000130592 G > A R187H; R249H; R253H; R256H; R377H WSU-DLCL2 MAST1 ENSG00000105613 G > A A74T DB MEF2B ENSG00000064489 T > A D83V DB MEF2C ENSG00000081189 A > G Y69H DB MEF2C ENSG00000081189 T > G E14A OCI-Ly1 MEF2C ENSG00000081189 T > G K5T OCI-Ly1 MKI67 ENSG00000148773 T > G K617N; K977N SU-DHL-6 MLL2 ENSG00000167548 C > A L3496L DB MLL2 ENSG00000167548 G > A Q2156* DB MLL2 ENSG00000167548 G > A S4824F NU-DHL-1 MLL2 ENSG00000167548 G > A R1323* OCI-Ly1 MLL2 ENSG00000167548 G > A Q3384* NU-DUL-1 MLL2 ENSG00000167548 C > A D635Y NU-DUL-1 NCKAP1L ENSG00000123338 A > G V105V OCI-Ly19 PCDHGC5 ENSG00000081853 A > G L726L WSU-DLCL2 PLCG2 ENSG00000197943 C > T G426G OCI-Ly7 PRDM15 ENSG00000141956 G > C L361V; L398V; L727V SU-DHL-6 PSAP ENSG00000197746 A > T L260H WSU-DLCL2 RBM39 ENSG00000131051 A > G I240T; I247T; I397T OCI-Ly7 RFTN1 ENSG00000131378 T > A H83L OCI-Ly1 RFXDC2 ENSG00000181827 C > T W685* NU-DUL-1 RNF14 ENSG00000013561 G > T Q133H; Q259H OCI-Ly1 SMG6 ENSG00000070366 G > A R767C OCI-Ly1 SOS2 ENSG00000100485 T > C S271G Karpas422 SPTBN1 ENSG00000115306 C > A D1318E; D1331E; D1344E DB STAT6 ENSG00000166888 T > C Q286R NU-DHL-1 STAT6 ENSG00000166888 C > G G375R OCI-Ly1 TNFAIP3 ENSG00000118503 G > A G367G DOHH-2 TP53 ENSG00000141510 G > A R155W; R248W DB TP53 ENSG00000141510 T > A K319* Karpas422 TP53 ENSG00000141510 T > C Y141C; Y234C SU-DHL-6 TP53 ENSG00000141510 A > C C83G; C176G OCI-Ly1 TP53 ENSG00000141510 C > T R65H; R158H OCI-Ly1 TP53 ENSG00000141510 C > T G152D; G245D OCI-Ly7 TP53 ENSG00000141510 C > T V50M; V143M NU-DUL-1 TSEN54 ENSG00000182173 C > T R490W OCI-Ly1 TSEN54 ENSG00000182173 G > C G525A OCI-Ly1 USP34 ENSG00000115464 T > A S1685S; S1837S SU-DHL-6 ZMYND8 ENSG00000101040 C > G V518L; V537L; V538L; V543L; V563L OCI-Ly7

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The invention claimed is:
 1. A method, comprising testing a sample from a human subject having B-cell non-Hodgkin lymphoma (NHL) for a mutation in Enhancer of Zeste Homolog 2 (EZH2), wherein the mutation in EZH2 is a non-synonymous substitution of Alanine (A) at position 682 (A682) of the wild-type EZH2 protein sequence and/or a non-synonymous substitution of Alanine (A) at position 692 (A692) of the wild-type EZH2 protein sequence.
 2. The method of claim 1, wherein testing the sample comprises detecting one or more mutations in a nucleic acid coding for Enhancer of Zeste Homolog 2 (EZH2).
 3. The method of claim 1, wherein testing the sample comprises detecting one or more mutations in a polypeptide coding for Enhancer of Zeste Homolog 2 (EZH2).
 4. The method of claim 1, wherein the sample is a tumor sample from a subject suspected of having B-cell non-Hodgkin lymphoma.
 5. The method of claim 1, wherein the substitution mutation is a somatic mutation.
 6. The method of claim 1, wherein the sample is a blood sample or a plasma sample.
 7. A method, comprising testing a sample from a human subject having B-cell non-Hodgkin lymphoma (NHL) for a mutation in Enhancer of Zeste Homolog 2 (EZH2), wherein the mutation in EZH2 is a non-synonymous substitution of Alanine (A) at position 682 (A682) of the wild-type EZH2 protein sequence, wherein the substitution mutation in Enhancer of Zeste Homolog 2 (EZH2) is A682G.
 8. The method of claim 7, wherein the sample is a tumor sample from a subject suspected of having B-cell non-Hodgkin lymphoma.
 9. The method of claim 7, wherein the substitution mutation is a somatic mutation.
 10. The method of claim 7, wherein the sample is a blood sample or a plasma sample.
 11. The method of claim 7, wherein the mutation in EZH2 further comprises a non-synonymous substitution of Alanine (A) at position 692 (A692) of the wild-type EZH2 protein sequence, wherein the substitution mutation is A692V.
 12. A method, comprising testing a sample from a human subject having B-cell non-Hodgkin lymphoma (NHL) for a mutation in Enhancer of Zeste Homolog 2 (EZH2), wherein the mutation in EZH2 is a non-synonymous substitution of Alanine (A) at position 692 (A692) of the wild-type EZH2 protein sequence, wherein the substitution mutation in Enhancer of Zeste Homolog 2 (EZH2) is A692V.
 13. The method of claim 12, wherein the sample is a tumor sample from a subject suspected of having B-cell non-Hodgkin lymphoma.
 14. The method of claim 12, wherein the substitution mutation is a somatic mutation.
 15. The method of claim 12, wherein the sample is a blood sample or a plasma sample.
 16. The method of claim 12, wherein the mutation in EZH2 further comprises a non-synonymous substitution of Alanine (A) at position 682 (A682) of the wild-type EZH2 protein sequence, wherein the substitution mutation is A682G. 